Uses and methods of il-2 superagonists, agonists, and fusions thereof

Abstract

Methods of treating a subject having cancer comprising administering an IL-2 mutein are provided.SOLUTION: CLAIMS What is claimed is: 1. A method of treating cancer, the method comprising administering a combination therapy comprising (i) anti-PD-1 antibodies or inhibitors, or anti-PD-L1 antibodies or inhibitors, and (ii) an IL-2 mutein comprising the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, wherein the numbering is according to the wild-type human IL-2 of the particular sequence.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. §119(e) to U.S. patent application Ser. No. 62 / 521,957, filed Jun. 19, 2017, and Ser. No. 62 / 679,687, filed Jun. 1, 2018, all of which are expressly incorporated herein by reference in their entireties. [Background technology]

[0002] Interleukin 2 (IL-2) is a pluripotent cytokine produced primarily by activated CD4+ T cells and plays an important role in the production of normal immune responses. IL-2 promotes the proliferation and expansion of activated T lymphocytes, enhances B cell growth, and activates monocytes and natural killer cells. These activities have led to the development of IL-2, which has been tested and approved for use as a 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 four alpha helical bundle type I cytokine that was first 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 promotes the development of regulatory T (Treg) cells (Cheng et al., Immunol Rev 241:63 (2011)), induces natural killer 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 acts 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 is expressed approximately 10 -8 K of M d It binds IL-2 at IL-2Rα, also known as the "low affinity" IL-2 receptor. IL-2 binding to cells expressing only IL-2Rα does not elicit a 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. Upon binding of IL-2 to IL-2Rα, this receptor sequentially triggers engagement of IL-2Rβ and IL-2Rγ, resulting in T cell activation. An IL-2 "superkine" with enhanced action due to its enhanced binding affinity for IL-2Rβ has previously been developed (Levin et al., 2013). Nature 484:529(2012)).

[0005] Despite the wealth of knowledge about IL-2, including IL-2 superagonists, there remains a need in the art for better combination therapies for the treatment of cancer, including combination therapy with anti-PD-1 antibodies and combination therapy with oncolytic viruses or CAR-T cells. The present invention fulfills this need and provides combination therapies of IL-2 superagonists or agonists for the treatment of cancer, in particular, combinations of IL-2 muteins containing substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type IL-2, with anti-PD-1 antibodies. Summary of the Invention

[0006] IL-2 has a wide range of effects on the immune system, playing an important role in regulating both immune activation and homeostasis. As an immune system stimulator, the IL-2 muteins of the present invention find use in combination with anti-PD-1 antibodies for the treatment of cancer.

[0007] In another aspect, provided herein are methods of treating a subject with cancer, comprising administering an IL-2 mutein in combination with an anti-PD-1 antibody or inhibitor. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any one of the IL-2 muteins disclosed herein. In some embodiments, the pharmaceutical composition comprises an IL-2 mutein with the amino acid substitutions L80F, R81D, L85V, I86V, and I92F.

[0008] Accordingly, in some embodiments, the invention provides methods of treating cancer comprising administering a combination therapy comprising: (i) an anti-PD-1 antibody or inhibitor; and (ii) an IL-2 mutein comprising the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, where the numbering is according to wild-type human IL-2 in SEQ ID NO: 2.

[0009] In some embodiments, the anti-PD-1 antibody or inhibitor is selected from the group consisting of nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and the PD-1 antibodies listed in U.S. Patent Publication No. 2017 / 0081409. In some embodiments, the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

[0010] In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprising an F42A substitution exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

[0011] In some embodiments, the IL-2 mutein further comprises a K43N substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprising a K43N substitution exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

[0012] In some embodiments, the IL-2 mutein further comprises a Y45A substitution, The sequence follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprising the Y45A substitution exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

[0013] In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprising an E62A substitution exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

[0014] In some embodiments, the IL-2 mutein is a fusion protein. In some embodiments, the fusion protein comprises an IL-2 as described above linked to albumin. In some embodiments, the fusion protein comprises an IL-2 as described above linked to an Fc antibody fragment. In some embodiments, the Fc antibody fragment is a human Fc antibody fragment. In some embodiments, the Fc antibody fragment comprises an N297A substitution.

[0015] In some embodiments, the cancer is selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, kidney cancer, liver cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and brain cancer. In some embodiments, the cancer is colon cancer.

[0016] In some embodiments, the IL-2 mutein exhibits increased binding ability to IL-2Rβ compared to wild-type human IL-2. In some embodiments, the IL-2 mutein exhibits higher binding affinity to IL-2Rβ compared to wild-type human IL-2.

[0017] The method of any one of claims 3 to 7, wherein the IL-2 mutein exhibits reduced binding affinity to CD25 compared to wild-type human IL-2.

[0018] In another aspect, provided herein is a pharmaceutical composition comprising any one of the IL-2 muteins or IL-2 mutein fusion proteins described herein and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition comprises an IL-2 mutein having the amino acid substitutions L80F, R81D, L85V, I86V, and I92F.

[0019] In some embodiments, the pharmaceutical composition comprises an anti-PD-1 antibody or inhibitor, any of the IL-2 muteins described herein, and a pharmaceutically acceptable carrier.

[0020] The present invention also provides an immune cell targeting or expression construct comprising an interleukin-2 receptor beta (IL-2Rβ) binding protein, wherein the equilibrium dissociation constant of said binding protein with IL-2Rβ is less than that of wild-type human IL-2 (hIL-2); linked to an immune cell targeting or expression construct comprising at least one other targeting moiety.

[0021] In some embodiments, the immune cell targeting or expression construct exhibits a cytotoxic effect on T cells, e.g., CD8+ T cells or CD4+ T cells.

[0022] In some embodiments, the construct is a chimeric antigen receptor (CAR), and the IL-2 mutein is fused to a transmembrane domain and linked to an intracellular signaling region. In some embodiments, the intracellular signaling region comprises a CD3 signaling domain. In some embodiments, the intracellular signaling region comprises one or more of a CD28 signaling domain, a CD137 signaling domain, an OX-40 signaling domain, an ICOS signaling domain, or a DAP10 signaling domain.

[0023] In some embodiments, the IL-2 mutein or other targeting moiety is fused to a ligand that binds to a protein associated with the TCR complex and is fused to a T cell receptor signaling domain polypeptide.

[0024] In some embodiments, the protein associated with the TCR complex is CD3.

[0025] In some embodiments, the T cell receptor signaling domain polypeptide comprises a CD4 cytosolic domain and a CD4 transmembrane domain.

[0026] In some embodiments, the construct is an antibody-binding T-cell receptor (ACTR) that comprises a chimeric antigen receptor component that binds with high affinity to a mutein of IL-2 or other targeting moiety.

[0027] In some embodiments, the CAR component comprises CD16 and an IL-2 mutein or other targeting moiety fused to an Fc sequence.

[0028] In some embodiments, the construct is a bispecific T cell exchanger (BiTE) comprising an IL-2 mutein fused to the variable region of an antibody that binds a component of the T cell receptor.

[0029] In some embodiments, the BiTE component of the T cell receptor is CD3.

[0030] In some embodiments, the IL-2 mutein contains the following amino acid substitutions, numbered according to wild-type hIL-2: L80F, R81D, L85V, I86V, and I92F.

[0031] In some embodiments, nucleic acids endocding the above constructs are provided.

[0032] In some embodiments, a vector comprising the nucleic acid is provided.

[0033] In some embodiments, a T cell is provided that comprises the above construct or vector. In some aspects, the T cell is CD4 + In some embodiments, the T cells are CD8 + T cells.

[0034] In some embodiments, an NK cell is provided that comprises the above-described construct or vector.

[0035] Also provided are isolated populations of the above immune cells.

[0036] Pharmaceutical formulations comprising the immune cell populations described above are also provided.

[0037] In some embodiments, methods are provided for targeting cells expressing IL-2 receptors, including cells expressing IL-2 receptors, comprising contacting the cells with a formulation comprising the immune cell population described above. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo.

[0038] The present invention also provides a method of treating cancer, comprising contacting an individual having cancer with an effective dose of a formulation comprising the above-described immune cell population.

[0039] In some embodiments, the cancer is leukemia, lymphoma, glioblastoma, medulloblastoma, breast cancer, head and neck cancer, renal cancer, ovarian cancer, Kaposi's sarcoma, acute myeloid leukemia, B-lineage malignancies, colorectal, pancreatic, renal, or mesothelioma.

[0040] The present invention also provides a method for targeting an IL-2 mutein protein to cancer cells, comprising contacting the cancer cells with an IL-2 mutein oncolytic virus combination, the combination comprising an IL-2 mutein conjugated to or expressed by an oncolytic virus, the oncolytic virus capable of targeting the cancer cells.

[0041] In some embodiments, the contacting occurs in vitro. In some embodiments, the contacting occurs in vivo.

[0042] In some embodiments, the oncolytic virus is selected from the group consisting of adenovirus, autonomously replicating alphavirus, vaccinia virus, Seneca Valley virus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus (VSV), herpesvirus (including HSV-1 and HSV-2), measles virus, poliovirus, reovirus, coxsackie virus, lentivirus, morbillivirus, influenza virus, Sinbis virus, myxoma virus, and retrovirus.

[0043] In some embodiments, the vaccinia virus genome comprises a thymidine kinase (TK) gene inactivated by an open reading frame excising a deletion of at least one nucleotide providing a replacement in the TK gene and / or a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding an IL-2 mutein as described herein.

[0044] In some embodiments, the in vivo contact results in an increase in the concentration of the IL-2 mutein protein in the tumor microenvironment compared to the concentration of the IL-2 mutein protein that is not conjugated to an oncolytic virus.

[0045] In some embodiments, the modified oncolytic viruses target the IL-2 mutein to immunosuppressive cells of the tumor microenvironment (TME), such as tumor-associated macrophages and MDSCs (myeloid-derived suppressor cells), to improve therapeutic benefit.

[0046] In some embodiments, the modified oncolytic virus targets the IL-2 mutein to one or more immunosuppressive cells that express one or more tumor antigens.

[0047] In some embodiments, the modified oncolytic virus targets the IL-2 mutein to the TME.

[0048] In some embodiments, the IL-2 mutein protein enhances effector T cells and / or NK cells.

[0049] In some embodiments, the IL-2 mutein suppresses Treg activity.

[0050] In some embodiments, the IL-2 comprises the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, the numbering of which is according to wild-type human IL-2 in SEQ ID NO:2.

[0051] According to the present invention, the vector comprises a vaccinia virus genome, the thymidine kinase gene of which is a replacement and / or partial replacement of the thymidine kinase gene (TK) gene. Also provided is a modified vaccinia virus vector, characterized in that the thymidine kinase gene is inactivated by an open reading frame that excises a deletion of at least one nucleotide providing a deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding an IL-2 mutein as described herein.

[0052] The present invention also provides a modified oncolytic adenovirus comprising (i) a modified nucleic acid, optionally with a deletion of nucleotides encoding amino acids 122-129 of the encoded E1A polypeptide, and (ii) an expression cassette comprising a polynucleotide encoding an IL-2 mutein as described herein.

[0053] In some embodiments, the IL-2 mutein directs the modified oncolytic virus to immunosuppressive cells of the tumor microenvironment (TME), such as tumor-associated macrophages and MDSCs (myeloid-derived suppressor cells), for improved therapeutic benefit.

[0054] In some embodiments, the IL-2 mutein protein directs the modified oncolytic virus to one or more tumor antigens.

[0055] In some embodiments, the IL-2 mutein protein directs the modified oncolytic virus to the TME.

[0056] In some embodiments, the IL-2 mutein protein enhances effector T cells and NK cells.

[0057] In some embodiments, the IL-2 mutein suppresses Treg activity.

[0058] The present invention also provides methods of treating cancer, comprising administering to a subject in need thereof an oncolytic virus capable of expressing an IL-2 mutein. In some aspects, the IL-2 mutein comprises the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, where the numbering is according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 oncolytic virus is selected from the group consisting of adenovirus, autonomously replicating alphavirus, vaccinia virus, Seneca Valley virus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus (VSV), herpesvirus (including HSV-1 and HSV-2), measles virus, poliovirus, reovirus, coxsackievirus, lentivirus, morbillivirus, influenza virus, Sinbis virus, myxoma virus, and retrovirus. [Brief explanation of the drawings]

[0059] [Figure 1]H9 synergizes with anti-PD-1 immunotherapy. The combination therapy produced robust responses in a dose-dependent manner. Anti-PD-1 antibody was administered intravenously at 10 mg / kg, three times every four days (10 mg / kg IV q4dx3). H9 (an IL-2 mutein with amino acid substitutions L80F, R81D, L85V, I86V, and I92F, whose numbering follows that of wild-type human IL-2 in SEQ ID NO: 2) was administered at the indicated doses of 5 μg q.d. or 25 μg q.d. (μg / mouse) according to the same dosing regimen. MC38 colon cancer model mice were then monitored for up to 40 days after tumor implantation. The combination of anti-PD-1 antibody and H9 increased the number of cures at both low and high doses, with a substantial increase at the 25 μg q.d. dose of H9. [Figure 2] Examples of IgG1, IgG2, IgG3, and IgG4 sequences are provided. [Figure 3] An exemplary H9-Fc fusion sequence is provided. [Figure 4] Comparative analysis of IL-13Rα1- and IL-13Rα2-specific IL-13 variants. Human IL-13 and IL-13Rα1- and IL-13Rα2-selective variant sequences are shown with the indicated residue numbers. Kinetic and affinity parameters were determined by surface plasmon resonance. [Figure 5] H9-Fc improved potency against key immune cells, and H9 and H9-Fc significantly improved potency against key effector T cells, particularly CD8+ T cells, involved in tumor cell killing. H9 and its Fc variants can significantly increase the relative activation of anti-tumor effector CD8+ T cells without losing potency against Tregs. [Figure 6]H9-Fc has similar in vivo efficacy and an enhanced PK profile compared to H9. An optimized dose and schedule for the enhanced PK variant of H9 were identified. H9-Fc enabled effective B16F10 tumor control on a biweekly schedule, a schedule similar to that of anti-PD-1 antibodies used in mice. Therefore, we predict weekly or biweekly administration of H9-Fc. Subcutaneous administration: Subcutaneous H9-Fc is an advantageous administration approach for future immunotherapeutic agents. Checkpoint inhibitors, proleukin, and competitive IL-2 therapies (NKTR-214, ALKS 4230) all require IV infusion, necessitating prolonged administration and monitoring times in the clinic. Subcutaneous administration offers a rapid and convenient administration that is commonly preferred by patients for commonly targeted cancer treatments. [Figure 7] Exemplary anti-PD-1 antibodies for use in the combinations of the present invention. [Figure 8] Exemplary anti-PD-L1 antibodies for use in the combinations of the invention. [Figure 9] Exemplary oncolytic viruses. DETAILED DESCRIPTION OF THE INVENTION

[0060] In order that the present disclosure may be more readily understood, certain terms and phrases are defined below throughout the specification.

[0061] 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 one of ordinary skill 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) provides those of skill 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.

[0062] As used herein, "IL-2" refers to wild-type IL-2, whether natural or recombinant. Mature human IL-2 occurs as a 133 amino acid sequence (less a signal peptide consisting of an additional 20 N-terminal amino acids) as described by Fujita et al., PNAS USA, 80, 7437-7441 (1983). The amino acid sequence of human IL-2 (SEQ ID NO: 1, full length) is available under Genbank accession locator N P_000577.2. The amino acid sequence of mature human IL-2 is shown in SEQ ID NO: 2 (human wild-type mature; substitution position numbering is based on this sequence). The mouse (Mus musculus) IL-2 amino acid sequence is found in Genbank accession locator (SEQ ID NO: 3). The amino acid sequence of mature mouse IL-2 is shown in SEQ ID NO: 4. SEQ ID NO: 1 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 2 APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT SEQ ID NO: 3 MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ SEQ ID NO:4 APTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDESATVVDFLRRWIAFCQSIISTSPQ

[0063] As used herein, "IL-2 mutein" refers to an IL-2 polypeptide that has specific substitutions relative to the interleukin-2 protein. IL-2 muteins are characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or other residues in the native IL-2 polypeptide chain. According to the present disclosure, all such insertions, deletions, substitutions, and modifications result in IL-2 muteins that retain IL-2Rβ binding activity. Exemplary muteins can include substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids.

[0064] Muteins also include conservative modifications and substitutions at other positions of IL-2 (i.e., those that have minimal effect on the secondary or tertiary structure of the 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 represent conservative changes: Group I: ala, pro, gly, gln, asn, ser, thr; Group II: cys, ser, tyr, thr; Group III: val, ile, leu, met, ala, phe; Group IV: lys, arg, his; Group V: phe, tyr, trp, his; and Group VI: asp, glu.

[0065] "Numbered according to IL-2" means identifying the selected amino acid by reference to the position where that amino acid normally occurs in the mature sequence of wild-type IL-2, e.g., R81 refers to the 81st amino acid, arginine, present in SEQ ID NO:2. L80 refers to the 80th amino acid, leucine, present in SEQ ID NO:2. L85 refers to the 85th amino acid, leucine, present in SEQ ID NO:2. I86 refers to the 86th amino acid, leucine, present in SEQ ID NO:2. I92 refers to isoleucine, the 86th amino acid present in SEQ ID NO: 2. I92 refers to isoleucine, the 92nd amino acid present in SEQ ID NO: 2. F42 refers to phenylalanine, the 42nd amino acid present in SEQ ID NO: 2. K43 refers to lysine, the 43rd amino acid present in SEQ ID NO: 2.

[0066] As used herein, the abbreviations for the genetically encoded L-enantiomeric amino acids used in the disclosed methods are conventional and are as follows in Table 1: [Table 1]

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

[0068] The term "cell type bearing the IL-2Rαβγ receptor" refers to cells known to have this receptor type, i.e., T cells, activated T cells, B cells, activated monocytes, and activated NK cells. The term "cell type bearing the IL-2Rβγ receptor" refers to cells known to have that receptor type, i.e., B cells, resting monocytes, and resting NK cells.

[0069] The term "identity" as used herein with respect to polypeptide or DNA sequences , refers to the subunit sequence identity between two molecules. If a subunit position in both molecules is occupied by the same monomeric subunit (i.e., the same amino acid residue or nucleotide), the molecules are identical at that position. The similarity between two amino acid or two nucleotide sequences is a direct function of the number of identical positions. Generally, sequences are aligned to obtain the highest order match. 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.

[0070] 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).

[0071] When variant IL-2 polypeptides of the disclosure are "substantially pure," they can be at least about 60% by weight (dry weight) of the polypeptide of interest, e.g., a polypeptide containing a variant IL-2 amino acid sequence. 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, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

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

[0073] A "partial agonist" is a compound that interacts with the same target as an agonist, but increasing doses of the partial agonist do not produce as great a biochemical and / or physiological effect as an agonist.

[0074] A "superagonist" (also called a "superkine") is a type of agonist that can produce a maximal response that is greater than the endogenous agonist of the target receptor, and therefore has an efficacy greater than 100%.

[0075] "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 (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). "Regulatory sequences" include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). See, e.g., Genedel (1990) in Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.). Regulatory sequences include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of a nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). Those skilled in the art will appreciate that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the expression level of the desired protein, and the like. Introduction of the expression constructs of the present invention into host cells to thereby express the human IL-2 mutein disclosed herein is also useful. 2 muteins or biologically active variants thereof can be produced.

[0076] 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 subsequent generations due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the terms used herein.

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

[0078] As used herein, the term "pharmaceutically acceptable carrier" includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics) can also be incorporated into the compositions.

[0079] As used herein, the term "anti-PD-1 antibody" refers to any antibody that binds to PD-1, including inhibitory antibodies. An "anti-PD-1 inhibitor" refers to an inhibitor that binds to and inhibits PD-1. Such anti-PD-1 antibodies and / or inhibitors include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475, among others.

[0080] As used herein, the terms "cancer" (or "cancerous"), "hyperproliferative," and "neoplastic" refer to cells with autonomous growth (i.e., an abnormal appearance or state characterized by rapidly proliferating cell growth). Hyperproliferative and neoplastic disease states may be classified as pathological (i.e., characterizing or constituting a disease state), or they may be classified as non-pathological (i.e., a deviation from normal but not associated with a disease state). The term is meant to include all types of cancerous growth or oncogenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. "Pathological hyperproliferative" cells occur in disease states characterized by malignant tumor growth. Examples of non-pathological hyperproliferative cells include the proliferation of cells associated with wound repair. The term "cancer" or "neoplasm" is used to refer to malignant tumors of various organ systems, including those affecting the lung, breast, thyroid, lymph glands and tissues, reproductive system, gastrointestinal tract, and genitourinary tract, as well as adenocarcinomas, which are generally considered to include malignant tumors such as most colon cancers, renal cell carcinoma, prostate and / or testicular cancer, non-small cell lung cancer, cancer of the small intestine, and cancer of the esophagus. Cancer generally includes 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.

[0081] The term "cancer" is art-recognized and refers to malignant tumors of epithelial or endocrine tissues, including respiratory system carcinoma, gastrointestinal system carcinoma, genitourinary system carcinoma, testicular carcinoma, breast carcinoma, prostate carcinoma, endocrine system carcinoma, and melanoma. "Adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0082] As used herein, the term "hematopoietic neoplastic disorder" refers to a disease involving hyperplastic / neoplastic cells of hematopoietic origin, e.g., arising from the myeloid, lymphoid, or erythroid lineages, or their precursor cells. Preferably, the disease is a poorly differentiated acute leukemia (e.g., erythroblastic leukemia and lymphoid malignancies include, but are not limited to, acute lymphoblastic leukemia (ALL), including B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy cell leukemia (HLL), and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphoma include, but are not limited to, non-Hodgkin's lymphoma and their variants, peripheral T-cell lymphoma, adult T-cell leukemia / lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease, and Reed-Stemberg disease.

[0083] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect can be preventative, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it partially or completely cures a disease and / or side effects caused by a disease. As used herein, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes the following: (a) preventing a disease from occurring in a subject who has been pre-diagnosed with the disease or who is at risk of acquiring the disease, but who has not yet been diagnosed with the disease; (b) suppressing the disease, i.e., preventing the development of the disease; and (c) palliating the disease, i.e., causing regression of the disease. A therapeutically effective amount can be an amount that reduces tumor number, tumor size, and / or increases survival.

[0084] The terms "individual," "subject," and "patient" are used interchangeably herein and refer to mammals, including, but not limited to, humans and non-human primates, including monkeys and humans, sport mammals (e.g., horses), livestock mammals (e.g., sheep, goats, etc.), pet mammals (dogs, cats, etc.), and rodents (e.g., mice, rats, etc.).

[0085] 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 not a biologically or otherwise undesirable material; e.g., the material can be administered to a subject without causing substantial undesirable biological effects. Thus, such pharmaceutical compositions can be used, for example, when administering an IL-2 mutein to a subject. In particular, an IL-2 mutein containing substitutions L80F, R81D, L85V, I86V, and I92F is administered to a subject with cancer in combination with an anti-PD-1 antibody. 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.

[0086] As used herein, the phrase "unit dosage form" refers to a physically discrete unit suitable as a unitary dosage for a subject to be treated, each unit containing a predetermined amount, optionally in association with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), that produces a desired effect (e.g., a prophylactic or therapeutic effect) when administered in one or more doses. 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.

[0087] In some embodiments, the unit dosage form may be, for example, a liquid composition, or a lyophilized or frozen composition. The compositions may be in ampoules or vials containing a syrup in a dry state, to which, for example, a sterile liquid carrier can be added prior to in vivo administration or delivery. Individual unit dosage forms may be included in multi-dose kits or containers. The IL-2 mutein in combination with an anti-PD-1 antibody and pharmaceutical compositions thereof may be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage.

[0088] The "therapeutically effective amount" falls within a relatively broad range that can be determined through experiments and / or clinical trials. For example, in the case of in vivo injection, for example, direct injection into the tissue or vasculature of a subject (e.g., liver tissue or vein). Other effective dosages can be easily determined by those skilled in the art through routine trials to establish dose-response curves.

[0089] An "effective amount" or "amount sufficient" refers to that amount, in single or multiple doses, 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), that provides a detectable response, a measurable or detectable degree, or an expected or desired result or benefit in a subject over any period of time (long or short term) (e.g., minutes, hours, days, months, years, or until a cure).

[0090] An "effective amount" or "sufficient amount" of a therapeutic dose (e.g., to provide an improvement or therapeutic benefit or refinement) is typically effective to provide a measurable response to one, more than one, or all adverse symptoms, consequences, or complications of a disease, e.g., one or more adverse symptoms, disorders, illnesses, pathologies, or complications caused by or associated with a disease, although reducing, alleviating, inhibiting, arresting, limiting, or controlling the progression or worsening of a disease would also be a satisfactory result. In some embodiments, an effective amount is an amount sufficient to reduce the number of tumors. In some embodiments, an effective amount is an amount sufficient to reduce the size of tumors. In some embodiments, an effective amount is an amount sufficient to increase survival rates.

[0091] "Prevention" and grammatical variations thereof refer to a method in which contact, administration, or in vivo delivery to a subject precedes the onset of disease. Administration or in vivo delivery to a subject can occur prior to the onset of adverse symptoms, conditions, complications, etc. caused by or associated with disease. For example, screening (e.g., genetic) can be used to identify such subjects as candidates for the described methods and uses, although the subjects may not manifest the disease. Thus, even if such subjects do not manifest symptoms of disease, such subjects include those who screen positive for an insufficient or defective amount of a functional gene product (protein) that leads to disease, or for the production of an abnormal, partially functional, or non-functional gene product (protein), as well as subjects who screen positive for an abnormal or defective (mutated) gene product (protein) that leads to disease.

[0092] I. Detailed Description Described herein are IL-2 muteins containing the substitutions L80F, R81D, L85V, I86V, and I92F, which have increased binding to the IL-2Rβ receptor and find use in combination therapy with anti-PD-1 antibodies. In some embodiments, the IL-2 muteins containing L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 (SEQ ID NO: 2; wild-type hIL-2), are designated H9. Such IL-2 muteins find use, for example, in combination with anti-PD-1 antibodies for the treatment of cancer. Also provided are nucleic acids encoding such IL-2 muteins, methods for making such IL-2 muteins, pharmaceutical compositions containing such IL-2 muteins, and therapeutic methods using such IL-2 muteins.

[0093] A. IL-2 muteins The substituted amino acid residue(s) may be, but are not necessarily, conservative substitutions, typically including substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; phenylalanine, tyrosine. These mutations may occur at amino acid residues that contact IL-2Rβ and / or IL-2Rγ.

[0094] More specifically, mutations (conservative or non-conservative, by addition(s) or deletion(s)) can be made at one or more positions. For example, mutations can be I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I. The sequences of exemplary IL-2 muteins are as follows: 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0095] In some embodiments, the substitutions in the IL-2 mutein include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further includes a F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further includes a Y45A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further includes an E62A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, Y45A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the substitutions in the IL-2 mutein include F42A, Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, substitutions in an IL-2 mutein include E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, substitutions in an IL-2 mutein include Y45A, E62A, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, substitutions in an IL-2 mutein include Y45A and E62A, numbered according to wild-type human IL-2 of SEQ ID NO: 2.

[0096] In some embodiments, substitutions in an IL-2 mutein that result in increased and / or enhanced IL-2Rβ binding include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, an IL-2 mutein for use in the present invention comprises L80F, R81D, L85V, I86V, and I92F and exhibits increased IL-2Rβ binding. In some embodiments, an IL-2 mutein for use in the present invention further comprises a substitution at position F42A. In some embodiments, an IL-2 mutein for use in the present invention further comprises a substitution at position K43N. In some embodiments, the mutein comprises one or more substitutions selected from the group consisting of substitutions L80F, R81D, L85V, I86V, and I92F, and F42A, Y45A, and E62A, all compared to wild-type human IL-2 (SEQ ID NO: 2).

[0097] In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include L80F, R81D, L85V, I86V, and I92F. In some embodiments, amino acid substitutions that increase IL-2Rβ binding affinity include L80F, R81D, L85V, I86V, and I92F.

[0098] In some embodiments, a subject IL-2 mutein having a higher binding affinity for IL-2Rβ compared to wild-type human IL-2 comprises the amino acid substitutions L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: TIFF2026001033000003.tif21170

[0099] In some embodiments, the IL-2 mutein is IL-2Rβ / IL-2Rγ cand (b) an increased ability to stimulate one or more signal transduction pathways that are dependent on heterodimerization. In some embodiments, a subject IL-2 mutein has an enhanced ability to stimulate STAT5 phosphorylation in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates STAT5 phosphorylation in IL-2Rβ+ cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or greater of the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, the IL-2 mutein stimulates STAT5 phosphorylation in IL-2Rβ+ cells by 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more, compared to the level at which wild-type IL-2 stimulates STAT5 phosphorylation in the same cells. In some embodiments, the IL-2Rβ+ cells are T cells. In specific embodiments, the T cells are CD8+ T cells. In some embodiments, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein comprises the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0100] In some embodiments, the mutein has an enhanced ability to stimulate ERK1 / ERK2 signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 signaling in IL-2Rβ+ cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level at which wild-type IL-2 stimulates pERK1 / ERK2 signaling in the same cells. In some embodiments, the IL-2 mutein stimulates pERK1 / ERK2 phosphorylation in IL-2Rβ+ cells by 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more, compared to the level at which wild-type IL-2 stimulates pERK1 / ERK2 phosphorylation in the same cells. In some embodiments, the IL-2Rβ+ cells are T cells. In specific embodiments, the T cells are CD8+ T cells. In some aspects, the CD8+ T cells are freshly isolated CD8+ T cells. In other embodiments, the CD8+ T cells are activated CD8+ T cells. In other embodiments, In some embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein contains the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0101] STAT5 and ERK1 / 2 signaling can be measured, for example, by phosphorylation of STAT5 and ERK1 / 2 using any suitable method known in the art. For example, STAT5 and ERK1 / 2 phosphorylation can be measured using antibodies specific for phosphorylated versions of these molecules in combination with flow cytometry analysis as described herein. In some embodiments, the mutein has enhanced ability to stimulate PI 3-kinase signaling in IL-2Rβ+ cells compared to wild-type human IL-2. In some embodiments, the IL-2 mutein stimulates PI 3-kinase signaling in IL-2Rβ+ cells at 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the level at which wild-type IL-2 stimulates PI 3-kinase signaling in the same cells. In some embodiments, the IL-2 mutein stimulates PI 3-kinase signaling in IL-2Rβ+ cells by 105%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195%, or more compared to the level at which wild-type IL-2 stimulates PI 3-kinase signaling in the same cells. In some embodiments, the IL-2Rβ+ cells are T cells. In specific embodiments, the T cells are CD8+ T cells. In some aspects, the CD8+ T cells are activated CD8+ T cells. In other embodiments, the IL-2Rβ+ cells are natural killer (NK) cells. In some embodiments, the IL-2 mutein contains the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2). PI3-kinase signaling can be measured using any suitable method known in the art. For example, PI3-kinase signaling can be measured using an antibody specific for phospho-S6 ribosomal protein in conjunction with flow cytometry analysis as described herein.

[0102] In some embodiments, the IL-2 mutein is a stimulator of IL-2 and / or IL-15 STAT5 phosphorylation in CD8+ T cells. In some embodiments, the mutein is a promoter of IL-2 and / or IL-15-induced CD8+ T cell proliferation. In some embodiments, the mutein is a stimulator of IL-2-dependent, TCR-induced cell proliferation. In some aspects, the IL-2 mutein contains the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0103] IL-2 promotes the differentiation of Th1, Th9, and Treg T cells and inhibits Th17 differentiation. Therefore, without being bound by a particular theory of operation, it is believed that IL-2 muteins that function as IL-2 superagonists can promote Th1, Th9, and / or Treg cell differentiation or inhibit Th17 cell differentiation. In some embodiments, the IL-2 mutein is a promoter of IL-2-dependent Th1, Th9, and / or Treg differentiation. In some embodiments, the mutein is an inhibitor of Th17 differentiation. In some aspects, the IL-2 mutein contains the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0104] In some embodiments, the IL-2 mutein signals less and / or independently of CD25 compared to wild-type human IL-2 (e.g., has reduced CD25 binding). In some embodiments, the mutein comprises one or more substitutions selected from the group consisting of substitutions L80F, R81D, L85V, I86V, and I92F, and F42A, Y45A, and E62A, all of which are compared to wild-type human IL-2 (SEQ ID NO: 2). In some embodiments, reduced and / or independent signaling for CD25 allows for preferential activation of effector T cells while limiting stimulation of Tregs. In some embodiments, reduced and / or independent signaling for CD25 allows for reduced toxicity. In some embodiments, the mutein comprises one or more substitutions selected from the group consisting of substitutions L80F, R81D, L85V, I86V, and I92F, and F42A, Y45A, and E62A, all of which are compared to wild-type human IL-2 (SEQ ID NO: 2).

[0105] In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells. In some embodiments, the IL-2 mutein is capable of increasing and / or restoring responsiveness to anergic NK cells within the tumor microenvironment. In some aspects, the IL-2 mutein comprises the substitutions L80F, R81D, L85V, I86V, and I92F compared to wild-type human IL-2 (SEQ ID NO: 2).

[0106] In some embodiments, the mutein is an inhibitor of IL-2-dependent activation of natural killer (NK) cells. IL-2 activation of NK cells can be measured by any suitable method known in the art, for example, by measuring IL-2-induced CD69 expression and / or cytotoxicity as described herein.

[0107] In some embodiments, the increase in IL-2Rβ binding affinity is any binding affinity to IL-2Rβ that is greater than the binding affinity of wild-type human IL-2 to IL-2Rβ, hi some embodiments, the binding affinity is a 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold, or more increase in binding affinity to IL-2Rβ compared to the binding affinity of wild-type human IL-2 to IL-2Rβ.

[0108] In some embodiments, the increased binding ability to IL-2Rβ is any binding ability to IL-2Rβ that is greater than the wild-type human IL-2 binding ability to IL-2Rβ, hi some embodiments, the binding ability is a 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold, or more increase in binding ability to IL-2Rβ compared to the wild-type human IL-2 binding ability to IL-2Rβ.

[0109] In some embodiments, subject IL-2 muteins that have higher binding affinity for IL-2Rβ compared to wild-type human IL-2 also exhibit reduced binding to CD25 and contain the amino acid substitutions F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduction in binding affinity is about 220-fold, i.e., from a Kd of about 6.6 nM for wild-type human IL-2 to about 1.4 μM for the mutein containing F42A, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: TIFF2026001033000004.tif20170

[0110] In some embodiments, the subject IL-2 muteins that have a higher binding affinity for IL-2Rβ compared to wild-type human IL-2 also exhibit reduced binding to CD25, The IL-2 muteins include the amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the reduced binding affinity is due to the availability of glycosylation at position 43 with the K43N substitution. Replacing asparagine with lysine (K43N) reduces and / or eliminates CD25 binding in IL-2 muteins containing the amino acid substitutions K43N, L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 muteins have the following amino acid sequence: TIFF2026001033000005.tif20170

[0111] In some aspects, the reduction in binding affinity to CD25 is any binding affinity to CD25 that is lower than wild-type human IL-2 binding affinity, hi some embodiments, the binding affinity is a 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 120-fold, 150-fold, 170-fold, 190-fold, 200-fold, 220-fold, 240-fold, or more decrease in binding affinity to CD25 compared to wild-type human IL-2 binding affinity to CD25.

[0112] In some embodiments, a subject IL-2 mutein having a higher binding affinity to IL-2Rβ and a lower binding affinity to CD25 compared to wild-type human IL-2 comprises the amino acid substitutions F42A, Y45A L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: TIFF2026001033000006.tif20170

[0113] In some embodiments, a subject IL-2 mutein having a higher binding affinity to IL-2Rβ and a lower binding affinity to CD25 compared to wild-type human IL-2 comprises the amino acid substitutions F42A, E62A L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: TIFF2026001033000007.tif20170

[0114] In some embodiments, a subject IL-2 mutein having a higher binding affinity to IL-2Rβ and a lower binding affinity to CD25 compared to wild-type human IL-2 comprises the amino acid substitutions F42A, Y45A, E62A L80F, R81D, L85V, I86V, and I92F. In some embodiments, the IL-2 mutein has the following amino acid sequence: TIFF2026001033000008.tif26170

[0115] In some embodiments, the IL-2 mutein sequence is SEQ ID NO: 2 or SEQ ID NO: 6. In some embodiments, the IL-2 mutein sequence is 90% identical to SEQ ID NO: 10 or any one of SEQ ID NO: 16. In some embodiments, the IL-2 mutein sequence is 95% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6-10. In some embodiments, the IL-2 mutein sequence is 98% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6-10. In some embodiments, the IL-2 mutein sequence is 99% identical to SEQ ID NO: 2 or any one of SEQ ID NO: 6-10.

[0116] Further exemplary IL-2 sequences are provided in the table below. [Table 2] TIFF2026001033000010.tif236170TIFF2026001033000011.tif81170

[0117] B. IL-2 Mutein Fusion Protein IL-2 muteins can be prepared as fusion or chimeric polypeptides comprising the subject IL-2 mutein and a heterologous polypeptide (i.e., a polypeptide that is not IL-2 or a variant thereof) (see, e.g., U.S. Patent No. 6,451,308). Exemplary heterologous polypeptides can increase the circulating half-life of the chimeric polypeptide in vivo, thus further enhancing the properties of the mutant IL-2 polypeptide. In various embodiments, the polypeptide that increases circulating half-life can be serum albumin, such as human serum albumin, PEG, a PEG derivative, or an Fc region of an IgG subclass of antibody lacking the IgG heavy chain variable region. Exemplary Fc regions can contain mutations that inhibit complement fixation and Fc receptor binding, or can be lytic, i.e., capable of binding complement or lysing cells via another mechanism, such as antibody-dependent complement lysis (ADCC; U.S. Patent No. 08 / 355,502, filed December 12, 1994).

[0118] An "Fc region" can be a naturally occurring or synthetic polypeptide homologous to the C-terminal domain of IgG produced by digesting IgG with papain. IgG Fc has a molecular weight of approximately 50 kDa. A mutant IL-2 polypeptide can include the entire Fc region or a smaller portion that retains the ability to extend the circulating half-life of the chimeric polypeptide of which it is a part. Furthermore, the full-length or 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 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.

[0119] In some embodiments, the IL-2 mutein is linked directly or indirectly to the heterologous fusion polypeptide.

[0120] 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: 16). In some embodiments, the linker is GGGGSGGGGGSGGGGGS (SEQ ID NO: 17). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). In some embodiments, the linker is GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 19).

[0121] 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. Therefore, the Fc receptor binding site can be disrupted by mutating or deleting Leu 235. For example, substituting Leu 235 with Glu inhibits the ability of the Fc region to bind to the high-affinity Fc receptor. The mouse C'1q binding site can be functionally disrupted by mutating or deleting Glu 318, Lys 320, and Lys 322 residues of IgG. For example, substituting Glu 318, Lys 320, and Lys 322 with Ala residues renders IgG1 Fc unable to direct 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 includes the Leu residue at position 235 of IgG Fc, and the C'1q binding site includes the Glu318, Lys320, and Lys322 residues of IgG1. Soluble IgG Fc has wild-type residues or conservative amino acid substitutions at these sites. Soluble IgG Fc can target cells for antibody-dependent cellular cytotoxicity or complement-directed cytolysis (CDC). Suitable mutations for human IgG are also known (see, for example, Morrison et al., The Immunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125, 1994).

[0122] In other embodiments, the chimeric polypeptide can comprise an IL-2 mutein of interest and a polypeptide that functions as an antigen tag, such as a FLAG sequence. The FLAG sequence is recognized by a biotinylated, highly specific 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.

[0123] In other embodiments, the chimeric polypeptide comprises a mutant IL-2 polypeptide and a heterologous polypeptide that functions to enhance expression or direct the cellular localization of the mutant IL-2 polypeptide, such as the Aga2p agglutinin subunit (e.g., Boder and Wittrup, Nature Biotechnol. 15:553-7, 1997).

[0124] In other embodiments, chimeric polypeptides can be produced that contain mutant IL-2 and an antibody or its antigen-binding portion. The antibody or antigen-binding component of the chimeric protein can serve as a targeting moiety. For example, it can be used to localize the chimeric protein to a specific cell subset or target molecule. Methods for producing cytokine-antibody chimeric polypeptides are described, for example, in U.S. Patent No. 6,617,135.

[0125] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody, or antigen-binding portion thereof, that disrupts the interaction between the PD-1 receptor and its ligand, PD-L1, and / or is an antibody to a component of the PD-1 / PD-L1 signaling pathway. Antibodies known in the art that bind to PD-1, disrupt the interaction of PD-1 with its ligand, PD-L1, and stimulate an anti-tumor immune response are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. For example, antibodies that target PD-1 and can be used in the present invention include, e.g., For example, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-1210 (ShangHai HengRui), human monoclonal antibody REGN2810 (cemiplimab, Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 Examples of suitable antibodies include, but are not limited to, the IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1, disrupts the interaction between PD-1 and PD-L1, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. For example, antibodies targeting PD-L1 and in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies that target PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the chimeras disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-PD-1 antibody.In some embodiments, the chimeric polypeptide comprises a fusion to an anti-PD-L1 antibody.

[0126] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CTLA-4 and disrupts its interaction with CD80 and CD86. Exemplary antibodies that target CTLA-4 include the FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently in human trials. Other suitable antibodies that target CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patent Nos. 6,984,720 and 6,682,7368, and U.S. Patent Application Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those skilled in the art will appreciate that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptides comprise a fusion to an anti-CTLA-4 antibody.

[0127] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody that targets LAG-3 is IMP321 (Immutep), which is currently in human trials. Other suitable antibodies that target LAG-3 are disclosed in U.S. Patent Application No. 2011 / 0150892, which is incorporated herein by reference. Those skilled in the art will appreciate that any antibody that binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-LAG-3 antibody.

[0128] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof targeting B7-H3 or B7-H4. While the B7 family does not have any defined receptors, these ligands are upregulated on tumor cells or tumor-infiltrating cells. An exemplary antibody targeting B7-H3 is MGA271 (Macrogenics), which is currently undergoing human trials. Other suitable antibodies targeting B7 family members are disclosed in U.S. Patent Application No. 2013 / 0149236, which is incorporated herein by reference. Those skilled in the art will appreciate that any antibody that binds to B7-H3 or H4 and stimulates an anti-tumor immune response is suitable for use in the chimeras disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-B7-H3 or B7-H4 antibody.

[0129] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets TIM-3 and disrupts its interaction with Galectin-9. Suitable antibodies that target TIM-3 are disclosed in U.S. Patent Application Publication No. 2013 / 0022623, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to TIM-3, disrupts its interaction with Galectin-9, and stimulates an anti-tumor immune response is suitable for use in the chimeric polypeptides disclosed herein. In some aspects, the chimeric polypeptide comprises a fusion to an anti-TIM-3 antibody.

[0130] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets 4-1BB / CD137 and disrupts its interaction with CD137L. Those skilled in the art will appreciate that antibodies that bind to 4-1BB / CD137, disrupt its interaction with CD137L or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-4-1BB / CD137 antibody.

[0131] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets GITR and disrupts its interaction with its ligand. Those skilled in the art will appreciate that antibodies that bind to GITR, disrupt its interaction with GITRL or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some aspects, the chimeric polypeptide comprises a fusion to an anti-GITR antibody.

[0132] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets OX40 and disrupts its interaction with its ligand. Those skilled in the art will appreciate that antibodies that bind to OX40, disrupt its interaction with OX40L or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-OX40 antibody.

[0133] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CD40 and disrupts its interaction with its ligand. One of skill in the art will appreciate that antibodies that bind to CD40, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD40 antibody.

[0134] In some embodiments, the chimeric polypeptide targets ICOS and its ligands The chimeric polypeptides disclosed herein include fusions to an antibody or antigen-binding portion thereof that disrupts the interaction with ICOS. Those skilled in the art will appreciate that antibodies that bind to ICOS, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptides include fusions to an anti-ICOS antibody.

[0135] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets CD28 and disrupts its interaction with its ligand. One of skill in the art will appreciate that antibodies that bind to CD28, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-CD28 antibody.

[0136] In some embodiments, the chimeric polypeptide comprises a fusion to an antibody or antigen-binding portion thereof that targets IFNα and disrupts its interaction with its ligand. Those skilled in the art will appreciate that antibodies that bind to IFNα, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the chimeric polypeptides disclosed herein. In some embodiments, the chimeric polypeptide comprises a fusion to an anti-IFNα antibody.

[0137] In some embodiments, the chimeric polypeptide comprises a fusion with a tumor antigen or a polypeptide that targets a tumor antigen. Generally, tumor antigens allow tumor cells to be distinguished from their normal cellular counterparts and may include, for example, tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). In some embodiments, tumor antigens are protooncogenes and / or tumor suppressors, as well as overexpressed or aberrantly expressed cellular proteins, tumor antigens produced by oncogenic viruses, carcinoembryonic antigens, altered cell surface glycolipids and glycoproteins, and / or cell type-specific differentiation antigens. Such tumor antigens may include melanoma antigens, cancer-testis antigens, epithelial tumor antigens, cell cycle regulatory proteins, prostate-specific antigens (including, for example, prostate carcinoma antigens as disclosed in U.S. Pat. No. 5,538,866), and lymphomas (U.S. Pat. Nos. 4,816,249; 5,068,177; and 5,227,159). Tumor antigens include, for example, HMW mucins bound to 2G3 and 369F10, c-erbB-2-related tumor antigen (a glycoprotein of approximately 42 kD or 55 kD), antigens of 40, 60, 100, and 200 kD bound to approximately 113F1, 9-O-acetyl GD3, p97, alpha-fetoprotein (AFP) (e.g., for germ cell tumors and / or hepatocellular carcinoma), carcinoembryonic antigen (CEA) (e.g., for intestinal, and sometimes lung, or breast cancer), CA-125 (e.g., for ovarian cancer), MUC-1 (e.g., for breast cancer), and IFN-γ (e.g., for ovarian cancer). These antigens may include, but are not limited to, tumor antigens (e.g., for cancer), epithelial tumor antigens (ETAs) (e.g., for breast cancer), tyrosinase (e.g., for malignant melanoma), melanoma-associated antigens (MAGEs) (e.g., for malignant melanoma), cancer / testis antigen 1 (CTAG1B), melanoma-associated antigen 1 (MAGEA1), aberrant Ras products, aberrant p53 products, overexpression of cyclins (including, e.g., cyclin B1), mutations in fibronectin, post-translational changes in MUC1 glycoprotein, secreted tumor antigens (including, e.g., gangliosides).

[0138] Other fusions may include fusions with pro-apoptotic payloads. Exemplary sequences are provided in the table below. In some embodiments, the IL-2 muteins described herein are fused to pro-apoptotic payloads, such as BAD, BAX, BAK, BIK, and / or BID sequences. In some embodiments, the pro-apoptotic payload is a Bcl-2 domain-containing peptide and / or a sequence of BAD, BAX, BAK, BIK, and / or BID sequences. Exemplary Pro-apoptotic Fusion The cases are provided in Table 3 below. [Table 3] In some specific embodiments, an IL-2 antagonist may be fused to a pro-apoptotic payload for the treatment of cancer. An "antagonist" is a compound that opposes the action of an agonist, for example, by preventing, reducing, inhibiting, or neutralizing the activity of the agonist. An "antagonist" is a compound that inhibits the activity of a target, e.g., a target of interest, even in the absence of an identified agonist. For example, constitutive activity of a target receptor can be prevented, inhibited, or reduced. Typically, IL-2 muteins with agonist or superagonist activity compared to wild-type IL-2 are used in the cancer treatment methods of the invention, although IL-2 muteins with antagonist properties can be used when such antagonists are fused to a pro-apoptotic payload. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions compared to wild-type IL-2 of SEQ ID NO:2: L18R, Q22E, Q126T, and S130R. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions compared to wild-type IL-2 of SEQ ID NO:2: L18R, Q22E, L80F, R81D, L85V, I86V, and Q126T. In some embodiments, the IL-2 antagonist comprises the following amino acid substitutions L18R, Q22E, L80F, R81D, L85V, I86V, Q126T, and S130R compared to the wild-type IL-2 of SEQ ID NO: 2. Exemplary antagonists that can be fused to a pro-apoptotic payload, such as those provided above, are provided below in Table 4. [Table 4A] TIFF2026001033000015.tif152170

[0139] Other fusions can also include fusions with anti-apoptotic payloads for use in prolonging the activation of CD8 cells, NK cells, and anergic NK cells, exemplary sequences of which are shown in the table below. Such prolonged activation of T cells can prove beneficial in cancer treatment therapy. [Table 4B] TIFF2026001033000017.tif225170TIFF2026001033000018.tif229170TIFF2026001033000019.tif112170

[0140] Other exemplary IL-2 fusions include those listed in the table below. [Table 5] TIFF2026001033000021.tif196170TIFF2026001033000022.tif196170TIFF2026001033000023.tif215170 TIFF2026001033000024.tif208170TIFF2026001033000025.tif223170TIFF2026001033000026.tif209170

[0141] In some embodiments, the IL-2 mutein-Fc fusion comprises one of the following sequences: [Table 6]

[0142] In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is 95% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is 95% identical to any one of SEQ ID NO:12-15 and / or SEQ ID NO:20-80 (e.g., any of the IL-2 sequences provided herein). In some embodiments, the IL-2 mutein sequence is 98% identical to any one of SEQ ID NO: 12 through SEQ ID NO: 15 and / or SEQ ID NO: 20 through SEQ ID NO: 80 (e.g., any of the IL-2 sequences provided herein).

[0143] C. IL-4, IL-13 for fusion with IL-2, IL-10, IL-12, IL-15, and IL-18 In some embodiments, the IL-2 mutein can be fused to an IL-4 mutein as described herein. In some embodiments, the IL-2 mutein can be fused to an IL-13 mutein as described herein. In some embodiments, the IL-2 mutein can be fused to IL-10. In some embodiments, the IL-2 mutein can be fused to IL-12. In some embodiments, the IL-2 mutein can be fused to IL-15. In some embodiments, the IL-2 mutein can be fused to IL-18. In some embodiments, such fusions function to specifically target cancer cells and / or cancer stem cells, reduce or inhibit cancer stem cell growth, and target immunosuppressive cells within the tumor microenvironment (TME).

[0144] Any IL-13 sequence or variant thereof may be used in fusion with the IL-2 muteins described herein. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. Exemplary IL-13 polypeptide sequences are provided in SEQ ID NOs:81-128, as well as in the table below. In some embodiments, the IL-13 polypeptide sequence is as provided in any one of SEQ ID NOs:81-128. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:81. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:82. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:83. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:84. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO:85. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 86. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 87. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 88. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 89. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 90. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 91. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 92. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 93. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 94. In some embodiments, the polypeptide sequence is SEQ ID NO: 95. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 96. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 97. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 98. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 99.In some embodiments, the polypeptide sequence is SEQ ID NO: 100. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 101. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 102. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 103. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 104. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 105. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 106. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 107. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 108. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 109. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 110. In some embodiments, the polypeptide sequence is SEQ ID NO: 111. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 112. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 113. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 114. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 115. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 116. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 117. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 118. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 119. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 120. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 121. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 122. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 123. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 124. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 125. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 126. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 127. In some embodiments, the IL-13 polypeptide sequence is SEQ ID NO: 128. IL-13 In some embodiments, the IL-13 polypeptide sequence is 90% identical to any one of SEQ ID NO: 81-128. In some embodiments, the IL-13 polypeptide sequence is 95% identical to any one of SEQ ID NO: 81-128. In some embodiments, the IL-13 polypeptide sequence is 98% identical to any one of SEQ ID NO: 81-128.In some embodiments, the IL-13 polypeptide sequence is 99% identical to any one of SEQ ID NO:81 through SEQ ID NO:128.

[0145] In some embodiments, any one of SEQ ID NOs: 81 through 128 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 81 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 82 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 83 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 84 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 85 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 86 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 87 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 88 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 89 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:90 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:91 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:92 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:93 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:94 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:94 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:96 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:97 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 98 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 99 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 100 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 101 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 102 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 103 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 104 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 105 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 106 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 107 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 108 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 109 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 110 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 111 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 112 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 113 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 114 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 115 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 116 is bound to an IL-2 or IL-2 mutein described herein.In some embodiments, SEQ ID NO: 117 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 118 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 119 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 120 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 121 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 122 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 123 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 124 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 125 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 126 is bound to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 127 is linked to IL-2 or an IL-2 mutein described herein. In some embodiments, SEQ ID NO: 128 is linked to IL-2 or an IL-2 mutein described herein. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO: 5; 5-2 SEQ ID NO: 6; 6-6 SEQ ID NO: 7; A2 SEQ ID NO: 8; B1 SEQ ID NO: 9; B11 SEQ ID NO: 10; C5 SEQ ID NO: 11; D10 SEQ ID NO: 12; E10 SEQ ID NO: 13; G8 SEQ ID NO: 14; H4 SEQ ID NO: 15; and H9 SEQ ID NO: 16.

[0146] In some embodiments, the IL-13 peptides of the present invention comprise the following amino acid substitutions: (1) L10F, L10I, L10V, L10A, L10D, L10T, L10H; (2) R11S, R11N, R11H, R11L, R11I; (3) I14L, I14F, I14V, I14M; (4) V18L, V18F, V18I; (5) E12A, (6) R65D, (7) R65D, (8) R65E, (9) R65F, (10) R65F, (11) R65F, (12) R65F, (13) R65F, (14) R65F, (15) R65F, (16) R65F, (17) R65F, (18) R65F, (19) R65F, (20) R65F, (21) R65F, (22) R65F, (23) R65F, (24) R65F, (25) R65F, (26) R65F, (27) R65F, (28) R65F, (29) R65F, (30) R65F, (31) R65F, (32) R65F, (33) R65F, (34) R65F, (35) R65F, (36) R65F, (37) R65F, (38) R65F, (39) R65F, (40) R65F, (41) R65F, (42) R65F, (43) R65F, (44) R65F, (45) R65F, (46) R65F, (47) R65 )R86K, R86T, R86M; (8) D87E, D87K, D87R, D87G, D87S; (9) T88I, T88K, T88R; (10) K89R, K89T, K89M; (11) L101 (12) K104R, K104T, K104M; (13) K105T, K105A, K105R, K105E; (14) F107L, F107I, F107V, F107M; and (15) R108K, R108T, R108M, which substitutions result in altered affinity for one or both of IL-13Rα1 and IL-13Rα2. In other embodiments, the altered residues are at two or more, three or more, four or more, five or more, but not more than 14 amino acids within the combined set of contact residues defined above. See International Patent Publication No. WO 2013 / 112871, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, amino acid substitutions include, but are not limited to, those provided in FIG.

[0147] The set of modifications can include the following specific modifications: (1) L10H; L10A; (2) R11L; (4) V18I; (7) R86M; R86K; R86T; (8) D87K; D87G; (9) T88R, T88S; T88K; (10) K89R; (11) L101N; (12) K104R; (13) K105A; K105E; (14) R108K. In some embodiments, the modification includes any one of the specific modifications listed. In some embodiments, the modification includes L10H. In some embodiments, the modification includes L10A. In some embodiments, the modification includes R11L. In some embodiments, the modification includes V18I. In some embodiments, the modification includes R86M. In some embodiments, the modification includes R86K. In some embodiments, the modification includes R86T. In some embodiments, the modification comprises D87K. In some embodiments, the modification comprises D87G. In some embodiments, the modification comprises T88R. In some embodiments, the modification comprises T88S. In some embodiments, the modification comprises T88K. In some embodiments, the modification comprises K89R. In some embodiments, the modification comprises L101N. In some embodiments, the modification comprises K104R. In some embodiments, the modification comprises K105A. In some embodiments, the modification comprises K105E. In some embodiments, the modification comprises R108K. In some aspects, the polypeptides comprising one or more modifications are conjugated to IL-2 or IL-2 muteins described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those provided in Figure 4. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0148] A particular set of modifications that increase the selectivity of binding to IL-13Rα2 versus IL-13Rα1 compared to the native IL-13 sequence include, but are not limited to: [L10D, R11I, V18I, R86K, D87K, k89R, R108K] (e.g., C2, e.g., SEQ ID NO: 31 or SEQ ID NO: 49) [L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, R108K] (e.g., C3, e.g., SEQ ID NO: 32 or SEQ ID NO: 50) [L10V, K89R, L101N, K105E, R108T] (e.g., C4, e.g., SEQ ID NO: 33 or SEQ ID NO: 31) [R11S, I14M, T88S, L101N, K105A, R108K] (e.g., C7, e.g., SEQ ID NO: 34 or SEQ ID NO: 52) [L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, R108K] (C9, e.g., SEQ ID NO: 53) [L10H, R86T, D87G, T88R, R108K] (C11, e.g., SEQ ID NO: 38 or SEQ ID NO: 55) [L10A, V18F, R86K, D87K, K89R, L101I, K104R, R108K] (D7, e.g., SEQ ID NO: 40 or SEQ ID NO: 57) ●[L10T / D;R11I;V18I;R86K;D87K / G;T88S;K89R;L101Y;K104R;K105T;R108K] ●[L10A / V;R86T;D87G;T88K;K89R;L101N;K104R;K105A / E;R108K / T]

[0149] In some embodiments, the set of modifications comprises L10V, K89R, L101N, K105E, R108T. In some embodiments, the set of modifications comprises R11S, I14M, T88S, L101N, K105A, and R108K (C7, e.g., SEQ ID NO: 35 or SEQ ID NO: 52). In some embodiments, the set of modifications comprises L10H, R11L, V18I, R86K, D87E, K89R, L101N, K105T, and R108K (C9, e.g., SEQ ID NO: 36 or SEQ ID NO: 53). In some embodiments, the set of modifications comprises L10H, R86T, D87G, T88R, and R108K (C11, e.g., SEQ ID NO: 38 or SEQ ID NO: 55). In some embodiments, the set of modifications comprises L10A, V18F, R86K, D87K, K89R, L101I, K104R, and R108K (D7, e.g., SEQ ID NO:40 or SEQ ID NO:57). In some embodiments, the set of modifications comprises L10T / D, R11I, V18I, R86K, D87K / G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10T, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R11I, V18I, R86K, D87K, T88S, K89R, L101Y, K104R, K105T, and R108K. In some embodiments, the set of modifications comprises L10D, R11I, V18I, R86K, D87G, T88S, K89R, L101Y, K104R, K105T, R108K. In some embodiments, the set of modifications comprises L10A / V, R86T, D87G, T88K, K89R, L101N, K104R, K105A / E, and R108K / T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K.In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108T. In some embodiments, the set of modifications comprises L10A, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108K. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105A, dR108T. In some embodiments, the set of modifications comprises L10V, R86T, D87G, T88K, K89R, L101N, K104R, K105E, and R108T. In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptide comprising one or more modifications is linked to IL-2 or an IL-2 mutein described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those provided in Figure 4. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0150] A particular set of modifications that increase the selectivity of binding to IL-13Rα1 versus IL-13Rα2 compared to the native IL-13 sequence include, but are not limited to: ●[L10V, V18I, D87S, D88S, L101F, K104R, K105T] ●[R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T] ●[L10V, V18I, D87S, T88S, L101F, K104R, K105T] ●[L10V / I;D87S;T88S;K89R;L101H / F;K104R;K105T] ●[L10I;V18I;R86T;D87G;T88S;K89R;L101Y / H;K104R;K105A] ●[L10V;V18I;D87S;T88S;L101F;K104R;K105T] ●[V18I, R86T, D87G, T88S, L101Y, K104R, K105A] ●[R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M] These substitutions are optionally combined with substitutions [E12A / G / S, R65D / E].

[0151] In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, and K105T. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, and K105T. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, and K105A. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, and K105T. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, and K105A. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, and F107M. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D / E.In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D / E.In some embodiments, the set of modifications is L10V, V18I. , D87S, T88S, L101F, K104R, K105T, E12S, and R65D / E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65D / E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D / E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65D. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65D.In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65D. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65D. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65D. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65D.In some embodiments, the set of modifications is V18I, R86T, D87G, T88S, L101Y, K104R, K105A,. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65D. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12A, and R65E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12A, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12A / G / S, and R65E.In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12G, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12G, and R65E. In some embodiments, the set of modifications comprises R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12G, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, D88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises R11S, V18I, R86K, D87G, T88S, K89M, L101Y, K104R, K105T, E12A / G / S, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises L10V / I, D87S, T88S, K89R, L101H / F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises L10I, V18I, R86T, D87G, T88S, K89R, L101Y / H, K104R, K105A, E12S, and R65E. In some embodiments, the set of modifications comprises L10V, V18I, D87S, T88S, L101F, K104R, K105T, E12S, and R65E. In some embodiments, the set of modifications comprises V18I, R86T, D87G, T88S, L101Y, K104R, K105A, E12S, and R65E.In some embodiments, the set of modifications includes R11I, V18I, R86K, D87G, T88S, L101H, K104R, K105A, F107M, E12S, and R65E. In some embodiments, the set of modifications includes L10V, E12A, V18I, R65D, D87S, T88S, L101F, K104R, and K105T (e.g., IL-13dn; see SEQ ID NO: 38). In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptide comprising one or more modifications is linked to IL-2 or an IL-2 mutein described herein. In some embodiments, the amino acid substitutions include, but are not limited to, those provided in Figure 4. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0152] A table of IL-13 sequences is shown below. [Table 7] TIFF2026001033000029.tif235170TIFF2026001033000030.tif236170TIFF2026001033000031.tif224170TIFF2026001033000032.tif210170 TIFF2026001033000033.tif210170TIFF2026001033000034.tif210170TIFF2026001033000035.tif236170TIFF2026001033000036.tif248170

[0153] Any IL-4 sequence or variant thereof may be used in combination with an IL-2 mutant, including those described herein. The IL-2 muteins may be used in fusion with a fusion protein or variant thereof. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. Exemplary polypeptide sequences are provided in SEQ ID NOs: 130-135, including any of those provided herein. In some embodiments, the IL-4 polypeptide sequence is as provided in any one of SEQ ID NOs: 130-135. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 130. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 131. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 132. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 133. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 134. In some embodiments, the IL-4 polypeptide sequence is SEQ ID NO: 135. In some embodiments, the IL-4 polypeptide sequence is 98% identical to any one of SEQ ID NOs: 130-135. In some embodiments, the IL-4 polypeptide sequence is 99% identical to any one of SEQ ID NOs: 130-135. In some embodiments, any one of SEQ ID NOs: 130-135 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 130 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 131 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 132 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 133 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 134 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 135 is linked to an IL-2 or IL-2 mutein described herein.In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0154] A table of IL-4 sequences is shown below. [Table 8] TIFF2026001033000038.tif129170

[0155] In some embodiments, the IL-2 mutein can be fused to an IL-10, IL-12, IL-15, and / or IL-18 sequence. In some embodiments, such a fusion functions to specifically target the fusion construct to NK cells and / or CD8+ cells. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, SEQ ID NO:136 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:137 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO:138 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 139 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 140 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, SEQ ID NO: 141 is linked to an IL-2 or IL-2 mutein described herein. In some embodiments, the IL-2 mutein can be fused to an IL-10, IL-12, IL-15, and / or IL-18 sequence as provided in SEQ ID NOs: 136-141 in the table below. [Table 9] TIFF2026001033000040.tif76170

[0156] Exemplary IL-2 mutein sequences include any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0157] In some embodiments, the cytokine-cytokine fusion is one of those included in the table below. [Table 10]

[0158] D. Recombinant Expression of IL-2 Muteins, Expression Vectors, and Host Cells In various embodiments, the polypeptides used in practicing the present invention are synthetic or produced by expression of recombinant nucleic acid molecules. Where the polypeptide is chimeric (e.g., a fusion protein comprising at least one mutant IL-2 polypeptide and a heterologous polypeptide), it may be encoded by a hybrid nucleic acid molecule comprising one sequence encoding all or part of an IL-2 mutein and a second sequence encoding all or part of a heterologous polypeptide. For example, the subject IL-2 muteins described herein may be fused to a hexahistidine tag, which facilitates purification of proteins expressed in bacteria, or a hemagglutinin tag, which facilitates purification of proteins expressed in eukaryotic cells.

[0159] Methods for constructing DNA sequences encoding IL-2 muteins and expressing those sequences in an appropriately transformed host include, but are not limited to, the use of PCR-assisted mutagenesis techniques. Mutations consisting of deletions or additions of amino acid residues to the 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 endonuclease. The resulting fragment can be expressed directly or further manipulated, for example, by ligating it 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. PCR-generated nucleic acids can also be used to produce a variety of mutant sequences.

[0160] 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. Each oligonucleotide usually contains a 5' or 3' overhang for complementary assembly.

[0161] In addition to producing mutant polypeptides through expression of nucleic acid molecules altered by recombinant molecular biology techniques, the subject IL-2 muteins can be chemically synthesized. Chemically synthesized polypeptides are routinely produced by those skilled in the art.

[0162] Once assembled (by synthesis, site-directed mutagenesis, or otherwise), the DNA sequence encoding the IL-2 mutein can be inserted into an expression vector and operably linked to expression control sequences suitable for expression of the IL-2 mutein in the desired transformed host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, 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 function in the selected expression host.

[0163] The DNA sequence encoding the 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 must be recognized by the cell selected for expression of the IL-2 mutein. It may be prokaryotic, eukaryotic, or a combination of the two. It may also be the native IL-2 signal sequence. 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 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.

[0164] E. Oncolytic Virus Targeting Sites In some examples, the IL-2 muteins described herein can be used to target oncolytic viruses (see, e.g., Allen et al., Mol. Ther. 16:1556-64, 2008). In some examples, oncolytic viruses can be used to target IL-2 muteins to tumors or TME. Many viruses can be used as oncolytic viruses, including adenoviruses and self-replicating alphaviruses, as well as oncolytic vaccinia viruses (see, e.g., WO2013038066, particularly Figure 17, the entire contents of which are incorporated herein by reference). Other oncolytic viruses include Seneca Valley virus, Newcastle disease virus (also known as Newcastle virus), Maraba virus, vesicular stomatitis virus (VSV), herpesviruses (including HSV-1), measles virus, poliovirus, reovirus, coxsackievirus, lentivirus, morbillivirus, influenza virus, sinbis virus, myxoma virus, and / or retroviruses (see, e.g., Twumasi-Boateng et al., "Oncolytic viruses as engineering platforms for combination immunotherapy"). (See, "Oncolytic Viruses," Nature Reviews Cancer, 2018, and Kaufman et al., Cancer Immunotherapy, 14:642-662 (2015), all of which are incorporated by reference in their entirety.) In some embodiments, oncolytic viruses include, but are not limited to, adenoviruses, self-replicating alphaviruses, vaccinia viruses, Seneca Valley viruses, Newcastle disease viruses, Maraba viruses, vesicular stomatitis viruses (VSVs), herpesviruses (including HSV-1 and HSV-2), measles viruses, polioviruses, reoviruses, coxsackieviruses, lentiviruses, morbilliviruses, influenza viruses, Sinbis viruses, myxoma viruses, and retroviruses. IL-2 superkines (H9 and IL-2 variants described herein) can also be used to target T cells / OVs to the TME. IL-2 variants (e.g., H9) can enhance effector T cells and NK cells, while IL-2 variants can suppress Treg activity. Other oncolytic viruses include, for example, oncoVex / T-VEC, which involves intratumoral injection of a replication-conditional herpes simplex virus that preferentially infects cancer cells. Alternatively, viruses engineered to express GM-CSF replicate within cancer cells, causing their lysis, releasing new viruses and a series of tumor antigens, secreting GM-CSF in the process. Such oncolytic virus vaccines enhance DC function in the tumor microenvironment to stimulate anti-tumor immune responses. These oncolytic viruses can be used to target or deliver the IL-2 muteins described herein to tumors. 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 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16.In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, the substitutions in the IL-2 mutein comprise L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutein described herein. In some embodiments, the oncolytic virus comprises a transgene capable of expressing an IL-2 mutein comprising the following amino acid substitutions, L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the oncolytic virus comprises a nucleic acid encoding an IL-2 mutein comprising the following amino acid substitutions, numbered according to wild-type human IL-2 of SEQ ID NO:2: L80F, R81D, L85V, I86V, and I92F. In some embodiments, the oncolytic virus comprises a transgene expressed as a therapeutic payload. In some embodiments, the therapeutic payload is IL-2 as described herein. In some embodiments, the therapeutic payload is an IL-2 mutein comprising the following amino acid substitutions, numbered according to wild-type human IL-2 of SEQ ID NO:2: L80F, R81D, L85V, I86V, and I92F.

[0165] In some embodiments, the oncolytic virus is an oncolytic vaccinia virus. In some embodiments, the oncolytic vaccinia virus vector is characterized in that the viral particle is an intracellular mature virus (IMV), an intracellular enveloped virus (IEV), a cell-associated enveloped virus (CEV), or an extracellular enveloped virus (EEV) type. In some embodiments, the oncolytic vaccinia virus particle is an EEV or IMV type. In some embodiments, the oncolytic vaccinia virus particle is an EEV type.

[0166] Generally, the vector is a vector that replicates preferentially in tumor cells and contains at least one transgene (e.g., a vector as described herein). The present invention also provides oncolytic vaccinia virus recombinants, cells, and pharmaceutical compositions containing the aforementioned vectors that express IL-2 muteins (IL-2 muteins described herein), promote antitumor effects and apoptosis induction, and modulate the host immune response in a subject. According to the present invention, oncolytic adenoviruses and oncolytic vaccinia viruses can be combined with IL-2 expression or targeting moieties described herein to target the oncolytic vaccinia virus or oncolytic adenovirus and / or express IL-2 muteins. Oncolysis releases tumor antigens and provides costimulatory danger signals. However, efficacy can be further improved by arming the virus. For example, CD40 ligand (CD40L, CD154) is known to induce apoptosis of tumor cells, which also triggers several immune mechanisms. One of these is the T helper type 1 (Thl) response, which leads to the activation of cytotoxic T cells and reduced immunosuppression. The present invention provides oncolytic viruses that express the IL-2 muteins of the present invention. In some embodiments, the present invention provides oncolytic viruses that are targeted (eg, "armed") with an IL-2 targeting moiety of the present invention.

[0167] In some embodiments, the oncolytic virus is a modified vaccinia virus vector, viral particle, host cell, pharmaceutical composition, and kit comprising a vaccinia virus genome, wherein the thymidine kinase gene is inactivated either by a substitution in the thymidine kinase (TK) gene and / or an open reading frame excising a deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a non-viral protein (e.g., an IL-2 mutein described herein that can be expressed). In another aspect, modified vaccinia virus vectors, viral particles, pharmaceutical compositions, or kits are provided that can be used for eliciting an immune response in a subject, for use in methods of inhibiting malignant cell growth in a mammal, for use in treating or preventing cancer, for detecting the presence of the modified vaccinia virus in a subject, and for cancer treatment, optionally as an in situ cancer vaccine in combination with adenovirus. In some embodiments, the present invention provides methods for producing a modified vaccinia virus comprising a vaccinia virus genome, wherein the thymidine kinase gene is inactivated by an open reading frame that excises a deletion of at least one nucleotide providing a replacement in the thymidine kinase (TK) gene and / or a partially deleted thymidine kinase gene, wherein the vaccinia growth factor gene is deleted, and wherein the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding a non-viral protein (e.g., an IL-2 mutein described herein), the method comprising the steps of providing a producer cell capable of sustaining production of vaccinia virus particles and carrying the modified vaccinia vector, culturing the producer cell under conditions suitable for viral replication and production, and harvesting the viral particles.

[0168] In some embodiments, the present invention provides methods of administering oncolytic viruses associated with or comprising nucleic acids encoding IL-2 muteins described herein, wherein the IL-2 muteins are expressed at the tumor location or expressed systemically in a subject. In some embodiments, the present invention also provides methods of administering oncolytic viruses "armed" or targeted with IL-2 muteins described herein. The route of administration will, of course, vary depending on the location and nature of the tumor and includes, for example, intradermal, transdermal, parenteral, intravenous, intramuscular, intranasal, subcutaneous, topical (e.g., in close proximity to the tumor, particularly using the vasculature or adjacent vasculature), transdermal, intratracheal, intraperitoneal, intraarterial, intravesical, intratumoral, inhalation, perfusion, lavage, and oral administration. Compositions are formulated for specific routes of administration.

[0169] 1. Oncolytic vaccinia virus Vaccinia virus is a member of the Orthopoxvirus genus in the Poxviridae family. It has a large double-stranded DNA genome (approximately 200 kb, approximately 200 genes), and a complex morphogenetic pathway produces infectious virions of different morphologies from each infected cell. The virus particle contains a lipid membrane(s) surrounding a core. The viral core contains viral structural proteins, a tightly packed viral DNA genome, and transcriptase. Vaccinia virus dimensions are approximately 360 x 270 x 250 nm and weigh approximately 5-10 fg. Genes are densely packed with small non-coding DNA fragments, and the open reading frame (ORF) lacks introns. Three classes of genes exist: early, intermediate, and late. Early genes (approximately 100 genes; immediate and late) primarily encode proteins related to immune regulation and viral DNA replication. The intermediate genes encode regulatory proteins required for expression of the late genes (e.g., transcription factors), and the late genes encode proteins required to make viral particles and enzymes that are packaged into new virions to initiate the next infection. Vaccinia virus replicates in the cytoplasm of the cell.

[0170] Various strains of vaccinia virus have been identified (including, by way of example, Copenhagen, modified virus Ankara (MVA), Lister, Tian Tan, Wyeth (= New York City Board of Health), and Western Reserve (WR)). The genome sequence of WR vaccinia has been determined (accession number AY243312). In some embodiments, the oncolytic vaccinia virus is Copenhagen, modified virus Ankara (MVA), Lister, Tian Tan, Wyeth, or Western Reserve (WR) vaccinia virus.

[0171] Different forms of virus particles have different roles in the viral life cycle. Several forms of virus particles exist: intracellular mature virus (IMV), intracellular enveloped virus (IEV), cell-associated enveloped virus (CEV), and extracellular enveloped virus (EEV). EEV particles have an extra membrane derived from the trans-Golgi network. This outer membrane has two important roles: a) it protects the internal IMV from immune attack, and b) it mediates the binding of the virus to the cell surface.

[0172] CEV and EEV are enveloped in host-derived membranes, which helps the virus evade host antibodies and complement. IMV and EEV particles have several differences in biological properties and play different roles in the viral life cycle. EEV and IMV bind to different (unknown) receptors (1), and they enter cells by different mechanisms. EEV particles enter cells via endocytosis, a pH-sensitive process. After internalization, the EEV outer membrane ruptures within acidified endosomes, exposing the IMV, which fuses with the endosomal membrane and releasing the viral core into the cytoplasm. On the other hand, IMV enters cells by fusion of the cellular and viral membranes, a pH-independent process. In addition, CEV induces the formation of actin tails from the cell surface, which propel the virion toward uninfected neighboring cells.

[0173] Furthermore, EEV is resistant to antibody (NAb) neutralization and complement toxicity, whereas IMV is not. Therefore, EEV mediates long-distance spread in vitro and in vivo. The comet inhibition test has become one method for measuring EEV-specific antibodies, because even if free EEV cannot be neutralized by EEV NAb, EEV release from infected cells is blocked by EEV NAb, and no comet-shaped plaques are observed. EEV has a higher specific infectivity (lower particle / pfu ratio) compared to IMV particles, making EEV an interesting candidate for therapeutic applications. However, the outer membrane of EEV is a very fragile structure, requiring careful handling of EEV particles, and obtaining the amount of EEV particles required for therapeutic applications is difficult. The EEV outer membrane ruptures at low pH (approximately pH 6). Once the EEV outer membrane ruptures, the virus particles within the envelope retain full infectivity as IMV.

[0174] Some host cell-derived proteins colocalize with EEV preparations but not with IEVs, and the amount of cellular proteins depends on the host cell line and virus strain. For example, WR EEV contains more cellular proteins than the IHD-J strain of VV. Host cell-derived proteins can modify the biological effects of EEV particles. For example, incorporation of the host membrane protein CD55 on the surface of EEV particles confers resistance to complement toxicity. In this study, we demonstrated that human A549 cell-derived proteins on the surface of EEV particles can target the virus to human cancer cells. A similar phenomenon was demonstrated in studies using human immunodeficiency virus type 1, where the host-derived ICAM-1 glycoprotein increased viral infectivity. The IEV membrane contains at least nine proteins, two of which are absent in CEV / EEV. The F12L and A36R proteins are involved in IEV transport to the cell surface; they are left behind and are not part of CEV / EEV (9, 11). Seven proteins, F13L, A33R, A34R, A56R, B5R, E2, and K2L, are common among IEV, CEV, and EEV. In the case of Western Reserve strains of vaccinia virus, typically up to 1% of viral particles are EEV, which are released into the culture supernatant prior to oncolysis of producer cells. 50-fold more EEV particles are released from the International Health Department (IHD)-J strain of vaccinia. However, IHD has not been studied for use in human cancer therapy. The IHD-W phenotype is primarily due to a point mutation within the A34R EEV lectin-like protein. Furthermore, deletion of A34R increases the number of EEV particles released. EEV particles can first be detected on the cell surface 6 hours after infection (as CEV) and in the supernatant 5 hours after infection (IHD-J strain). Infections with low multiplicities of infection (MOI) result in a higher proportion of EEV particles compared to high viral loads. The balance between CEV and EEV is influenced by the host cell and the virus strain.

[0175] Vaccinia was used to eradicate smallpox and has since been used as an expression vector for foreign genes and as a live recombinant vaccine for infectious diseases and cancer. Because vaccinia virus is the most widely used poxvirus in humans, safety data for its use in humans is extensive. Hundreds of thousands of people have been safely vaccinated with modified vaccinia virus strains during smallpox vaccination programs worldwide, with very rare serious adverse events reported: systemic vaccinia (systemic spread of vaccinia within the body), erythema multiforme (toxic / allergic reaction), eczema vaccinatum (widespread infection of the skin), progressive vaccinia (tissue destruction), and post-vaccinial encephalitis.

[0176] Between the 1960s and 1990s, 44 melanoma patients were treated in early clinical trials with wild-type vaccinia virus, with an overall objective response rate of 50% for the injected tumors and some beneficial immunological responses (36). Wild-type vaccinia virus has also been used to treat bladder cancer, lung cancer, kidney cancer, and myeloma, with only mild adverse events. JX-594, an oncolytic Wyeth strain vaccinia virus encoding GM-CSF, has been successfully evaluated in three phase I trials, and preliminary results from a randomized phase II trial have been presented at a scientific meeting.

[0177] Vaccinia virus is attractive for cancer gene therapy due to several characteristics. It has a natural tropism toward cancer cells, and deleting portions of the viral genes can significantly enhance selectivity. The present invention relates to the use of a double-deleted vaccinia virus (vvdd) in which two viral genes, viral thymidine kinase (TK) and vaccinia growth factor (VGF), are at least partially deleted. The TK and VGF genes are required for viral replication in normal cells but not in cancer cells. Partial TK deletions can be engineered in the TK region that confer activity.

[0178] TK-deficient vaccinia viruses rely on cellular nucleotide pools present in dividing cells for DNA synthesis and replication. In some embodiments, TK deletion significantly limits viral replication in quiescent cells, allowing efficient viral replication to occur only in actively dividing cells (e.g., cancer cells). VGF is secreted from infected cells and has a paracrine priming effect on surrounding cells by acting as a mitogen. The replication of VGF-deficient vaccinia viruses is highly attenuated in quiescent (non-cancer) cells. The effects of TK and VGF deletion have been shown to be synergistic.

[0179] 2. Oncolytic adenovirus Generally, adenoviruses are 36 kb linear, double-stranded DNA viruses (Grunhaus and Horwitz, 1992). The term "adenovirus" or "AAV" includes AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3 (AAV3), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV9_hu14, avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to an AAV that can infect primates, "non-primate AAV" refers to an AAV that can infect non-primate mammals, and "bovine AAV" refers to an AAV that can infect bovine mammals.

[0180] Adenoviral infection of host cells results in episomal maintenance of adenoviral DNA, thereby reducing the potential genotoxicity associated with vector integration. Adenoviruses are also structurally stable, and no genome rearrangements have been detected after extensive amplification. Adenoviruses can infect virtually all epithelial cells, regardless of their cell cycle stage. (See, e.g., US 2006 / 0147420, incorporated herein by reference in its entirety.) Furthermore, the adenoviral E1a and E4 regions are essential for efficient and productive infection of human cells. The E1a gene is the first viral gene transcribed during productive infection, and its transcription is independent of the action of any other viral gene products. However, expression of the E1a gene is required for transcription of the remaining early viral genes. In addition to regulating E1a gene expression, the E1a promoter integrates signals for viral genome packaging as well as sites required for the initiation of viral DNA replication. Schmid, S.I., and Hearing, P., in Current Topics in Microbiology and Immunology, vol. 199:pages 67-80 (1995).

[0181] In some embodiments, the oncolytic virus is an oncolytic adenovirus. It has been established that naturally occurring viruses can be engineered to produce oncolytic effects in tumor cells (Wildner, 2001; Jacotat, 1967; Kim, 2001; Geoerger et al., 2002; Yan et al., 2003; Vile et al., 2002, each of which is incorporated herein by reference). In the case of adenovirus, specific deletions in the adenovirus genome can weaken its ability to replicate in normal quiescent cells, while retaining its ability to replicate in tumor cells. One such conditionally replicating adenovirus, Δ24, was described by Fueyo et al. (2000) and is also referenced in U.S. Patent Application No. 2003 / 0138405, each of which is incorporated herein by reference. Δ24 adenovirus is derived from adenovirus type 5 (Ad-5) and contains a 24-base pair deletion in the CR2 portion of the E1A gene. See, for example, WO2001 / 036650A2, which is incorporated herein by reference in its entirety.

[0182] Oncolytic adenoviruses are conditionally replicating adenoviruses (CRAs) such as Delta24. D), which possess several properties that make them candidates for use as biotherapeutics. One such property is the ability to replicate in permissive cells or tissues, which amplifies the original oncolytic viral input and aids in the spread of the drug to neighboring tumor cells, where it exerts a direct antitumor effect.

[0183] In some embodiments, the oncolytic component of Delta24 is coupled with a transgene expression approach to generate armed Delta24. Armed Delta24 adenoviruses can be used to generate or enhance bystander effects within tumors and / or to generate or enhance detection / imaging of oncolytic adenovirus in patients or tumor-associated tissues and / or cells. In some embodiments, combining oncolytic adenoviruses with various transgene strategies (e.g., expression of IL-2 muteins) improves potential therapeutic capabilities, including potential for various refractory tumors, and provides improved imaging capabilities. In certain embodiments, oncolytic adenoviruses can be administered with replication-deficient adenoviruses, another oncolytic virus, replication-competent adenoviruses, and / or wild-type adenoviruses, each of which can be administered simultaneously with, before, or after the other adenoviruses.

[0184] In some embodiments, the E1a adenoviral vector comprises a basic adenoviral E1a promoter, including a CAAT box, a TATA box, and a start site for transcription initiation, replaced with a basic promoter that exhibits tumor specificity and is preferably E2F-responsive, more preferably the human E2F-1 promoter. Thus, the virus is suppressed in cells lacking or in which molecules that activate transcription from E2F-responsive promoters are nonfunctional. Normal, non-dividing or quiescent cells, classified as transcription factors, bind pRb or retinoblastoma protein, rendering E2F unavailable to bind and activate E2F-responsive promoters. In contrast, cells containing free E2F should support E2F-driven transcription. One example of such cells is a tumor cell lacking pRb function, allowing productive viral infection to occur. In some embodiments, the E1a adenoviral vector is targeted using the IL-2 moiety described herein.

[0185] Retention of the enhancer sequence, packaging signal, and DNA replication initiation site in the E1a promoter ensures that adenoviral infection proceeds to wild-type levels in neoplastic cells lacking pRb function. Essentially, the modified E1a promoter confers tumor-specific transcriptional activation, resulting in substantial tumor-specific killing, but with enhanced safety in normal cells.

[0186] In some embodiments, an E1a adenoviral vector is prepared by replacing the endogenous E1a promoter with an E2F-responsive promoter, leaving intact the elements upstream of nucleotide 375 of the adenovirus 5 genome. The nucleotide numbering is as described, for example, by Schmid, S.I., and Hearing, P. Current Topics in Microbiology and Immunology, vol. 199: pages 67-80 (1995). This includes all seven A repeat motifs identified for viral genome packaging. The sequence from nucleotide 375 to nucleotide 536 is deleted via the BsaAI to BsrBI restriction start site, while still retaining 23 base pairs upstream of the translation initiation codon for the E1A protein. Using known materials and methods, an E2F-responsive promoter, preferably human E2F-1, is used in place of the deleted endogenous E1a promoter sequence. The E2F-1 promoter can be isolated as described in Example 1.

[0187] The E4 region is involved in many events occurring late in adenovirus infection and is required for efficient viral DNA replication, late mRNA accumulation and protein synthesis, splicing, and shutting off host cell protein synthesis. Adenoviruses lacking most of the E4 transcription unit are severely replication-defective and generally require propagation in E4-complementing cell lines to achieve high titers. The E4 promoter is located near the right end of the viral genome and directs transcription of multiple open reading frames (ORFs). Many regulatory elements important for mediating maximal transcriptional activity have been characterized in this promoter. In addition to these sequences, the E4 promoter region contains regulatory sequences required for viral DNA replication. A depiction of the E4 promoter and the location of these regulatory sequences can be seen in Figures 2 and 3 of U.S. Patent No. 7,001,596, which is incorporated herein by reference in its entirety.

[0188] In some embodiments, the adenoviral vector has an E4 basal promoter substituted with an E2F-responsive promoter, preferably the human E2F-1 promoter, which has been demonstrated to exhibit tumor specificity. The reasons for the preference for an E2F-responsive promoter to drive E4 expression are the same as those discussed above in the context of E1a adenoviral vectors in which the E1a promoter is substituted with an E2F-responsive promoter. The tumor suppressor function of pRb correlates with its ability to repress E2F-responsive promoters, such as the E2F-1 promoter (Adams, P.D., and W.G. Kaelin, Jr. 1995, Cancer Biol. 6:99-108; Sellers, W.R., and W.G. Kaelin, Jr. 1996, Biochim Biophys Acta). Errata published in 1996 Dec. 9;1288(3):E-1, Biochim Biophys Acta. 1288:M1-5. Sellers, W. R., J. W. Lodgers, and W. G. Kaelin, Jr. 1995, Proc Natl Acad Sci USA. 92:11544-8. The human E2F-1 promoter has been extensively characterized and shown to be responsive to the pRb signaling pathway, including pRb / p107, E2F-1 / -2 / -3, and G1 cyclin / cdk complexes, as well as E1A (Johnson, DG, K. Ohtani, and J.R. Nevins. 1994, Genes Dev. 8:1514-25; Neuman, E., E.K. Flemington, W.R. Sellers, and W.G. Kaelin, Jr. 1995, Mol. Cell Biol. 15:4660; Neuman, E., W.R. Sellers, J.A.M. McNeil, J.B. Lawrence, and W.G. Kaelin, Jr. 1996, Gene. 173:163-9). Most, if not all, of this regulation is due to the presence of multiple E2F sites within the E2F-1 promoter. Therefore, viruses carrying this(these) modification(s) are expected to be attenuated in normal cells containing an intact (wild-type) pRb pathway, but to exhibit a normal infection / replication profile in cells lacking the inhibitory function of pRb.To maintain the normal infection / replication profile of this mutant virus, we retained the inverted terminal repeats (ITRs) at the distal end of the E4 promoter, because they contain all the regulatory elements necessary for viral DNA replication (Hatfield, L. and P. Hearing. 1993, J. Virol. 67:3931-9; Rawlins, D.R., P.J. Rosenfeld, R.J. Wides, M.D. Challberg, and T.J. Kelly, Jr. 1984, Cell. 37:309-19; Rosenfeld, P.J., E.A.O. Neill, R.J. Wides, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:875-86; Wides, R.J., M.D. Challberg, D.R. Rawlins, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:864-74). This facilitates the acquisition of wild-type levels of virus in pRb pathway-deficient tumor cells infected with this virus.

[0189] In some embodiments, the E4 promoter is located near the right end of the viral genome, and it directs transcription of multiple open reading frames (ORFs) (Freyer, G.A., Y. Katoh, and 1984, Nucleic Acids Res. 12:3503-19; Tigges, M.A., and H.J. Raskas. 1984. Adenovirus 2 early region 4 mRNA splice junctions: multiple splice sites generate 18-24 RNAs. J. Virol. 50:106-17; Virtanen, A.P. Gilardi, A. Naslund, J.M. LeMoullec, U. Pettersson, and M. Perricaudet. 1984, J. Virol. 51:822-31). Many regulatory elements that mediate transcriptional activity have been characterized in this promoter (Berk, A. J. 1986, Annu Rev Genet. 20:45-79; Gilardi, P., and M. Perricaudet. 1986, Nucleic Acids Res. 14:9035-49; Gilardi, P., and M. Perricaudet. 1984, Nucleic Acids Res. 12:7877-88; Hanaka, S., T. Nishigaki, PA Sharp, and H. Handa. 1987, Mol Cell Biol. 7:2578-87; Jones, C., and K. A Lee. 1991, Mol Cell Biol. 11:4297-305; Lee, K. A., and M. R. Green. 1987, Embo J. 6:1345-53).In addition to these sequences, the E4 promoter region contains elements involved in viral DNA replication (Hatfield, L., and P. Hearing. 1993, J. Virol. 67:3931-9; Rawlins, D.R., P.J. Rosenfeld, R.J. Wides, M.D. Challberg, and T.J. Kelly, Jr. 1984, Cell. 37:309-19; Rosenfeld, P.J., E.A.O. Neill, R.J. Wides, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:875-86; Wides, R.J., M.D. Challberg, D.R. Rawlins, and T.J. Kelly. 1987, Mol. Cell. Biol. 7:864-74). A depiction of the E4 promoter and the location of these regulatory sequences can be seen in Figures 1 and 2. See also Jones, C., and KALee. Mol Cell Biol. 11:4297-305 (1991). With these considerations in mind, the E4 promoter shuttle was designed by creating two novel restriction endonuclease sites: an XhoI site at nucleotide 35,576 and an SpeI site at nucleotide 35,815 (see Figure 3). Digestion with both XhoI and SpeI removes nucleotides 35,581 to 35,817. This effectively removes bases -208 to +29 relative to the E4 transcription start site, including all sequences shown to have the greatest effect on E4 transcription. Notably, this encompasses the two inverted repeats of the E4F binding site, which have been demonstrated to have the most significant effect on promoter activation. However, all three Sp1 binding sites, two of the five ATF binding sites, and both the NF1 and NFIII / Oct-1 binding sites, which are important for viral DNA replication, are retained.

[0190] In some embodiments, the E2F-responsive promoter is the human E2F-1 promoter. The key regulatory elements in the E2F-1 promoter that mediate response to the pRb pathway have been mapped both in vitro and in vivo (Johnson, DG, K. Ohtani, and JR Nevins. 1994, Genes Dev. 8:1514-25; Neuman, E., EK Flemington, WR Sellers, and WG Kaelin, Jr. 1995, Mol Cell Biol. 15:4660; Parr, MJ, Y. Manome, T. Tanaka, P. Wen, DW Kufe, WG Kaelin, Jr., and H A Fine. 1997, Nat Med. 3:1145-9). Therefore, we have isolated a human E2F-1 promoter fragment from base pairs -218 to +51 relative to the transcription start site, as described in Spe. The vector was isolated by PCR using primers that incorporated I and XhoI sites into the E4 promoter shuttle, creating the same sites present in the E4 promoter shuttle and allowing direct replacement of the E4 promoter with the E2F-1 promoter.

[0191] F. Nucleic Acid Molecules Encoding Variant IL-2 In some embodiments, a subject IL-2 mutein, alone or as part of a chimeric polypeptide as described above, can be obtained by expression of a nucleic acid molecule. As an IL-2 mutein can be described in terms of its identity to a wild-type IL-2 polypeptide, the nucleic acid molecule encoding it necessarily has a certain identity to a nucleic acid molecule encoding wild-type IL-2. For example, a nucleic acid molecule encoding a subject IL-2 mutein 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: 2).

[0192] The provided nucleic acid molecules can include naturally occurring sequences or sequences that differ from those occurring in nature but encode the same polypeptide due to the degeneracy of the genetic code. These nucleic acid molecules can be composed of RNA or DNA (e.g., genomic DNA, cDNA, or synthetic DNA such as that produced by phosphoramidite-based synthesis), or combinations or modifications of nucleotides within these types of nucleic acids. Furthermore, nucleic acid molecules can be double-stranded or single-stranded (i.e., either the sense strand or the antisense strand).

[0193] Nucleic acid molecules are not limited to sequences encoding polypeptides, but can also include some or all of the non-coding sequences upstream or downstream of the coding sequence (e.g., the coding sequence for IL-2).Those skilled in the art of molecular biology are familiar with routine procedures for isolating nucleic acid molecules.They can be produced, for example, by treating genomic DNA with restriction endonucleases or by performing polymerase chain reaction (PCR).If the nucleic acid molecule is ribonucleic acid (RNA), the molecule can be produced, for example, by in vitro transcription.

[0194] Exemplary isolated nucleic acid molecules of the present disclosure can include fragments not found as such in nature. Thus, the present disclosure encompasses recombinant molecules, such as those in which a nucleic acid sequence (e.g., a sequence encoding a mutant IL-2) is incorporated into a vector (e.g., a plasmid or viral vector) or into the genome of a heterologous cell (or the genome of a homologous cell at a location other than the natural chromosomal location).

[0195] As noted above, the subject IL-2 muteins can be present as part of a chimeric polypeptide. In addition to, or instead of, the heterologous polypeptides described above, the subject nucleic acid molecules can 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 , G418r ), dihydrofolate reductase (DHFR), hygromycin B-phosphotransferase (HPH), thymidine kinase (TK), lacz (encoding β-galactosidase), and xanthine guanine phosphoribosyltransferase (XGPRT). Those of skill in the art will recognize additional useful reagents, e.g., additional sequences that can serve as markers or reporters.

[0196] The subject nucleic acid molecules can be obtained by introducing mutations into DNA encoding IL-2 obtained from any organism's cells, such as mammalian cells. Thus, the subject nucleic acids (and the polypeptides they encode) can be from mice, rats, guinea pigs, cows, sheep, horses, pigs, rabbits, monkeys, baboons, dogs, or cats. In one embodiment, the nucleic acid molecules are human.

[0197] G. Chimeric Antigen Receptor (CARS) Targeted immunotherapy has emerged as a promising area of ​​research in the treatment of malignant tumors and has attracted significant interest in recent years. Indeed, treatment of lymphoma patients using recombinant or genetically modified T cells targeting CD19 malignant cells has been reported. This has led to increased attention being paid to antigens present on cancer cells as targets for gene therapy and immunotherapy. These CARS can be used to target or deliver the IL-2 muteins described herein to tumors, or even enable systemic IL-2 mutein expression. 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 90% identical to any one of SEQ ID NO:2, SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO: 16. In some embodiments, the substitutions in the IL-2 mutein comprise L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 in SEQ ID NO:2.

[0198] Genetic engineering of autologous or allogeneic T cells or NK cells to specifically target specific tumor antigens offers a strategy to circumvent the failure of most tumor cells to induce cytotoxic immune responses. In some embodiments, these engineered T cells or NK cells can be used to target the IL-2 muteins described herein to tumors, for example, so that the IL-2 muteins are expressed at the tumor site. These techniques are based on genetic modification of human immune cells, which can be extracted from patients or donors by leukapheresis. Specific cells, usually T cells, are purified and engineered to express receptors that target the cancer antigen of interest. Engineering may utilize transduction by retroviruses, lentiviruses, transposons, mRNA electroporation, etc. The immune cells can be expanded to the desired dose and introduced into the patient. The engineered cells can specifically kill cancer cells through cell-mediated toxicity (cytotoxic T cells) and / or by eliciting an immune response against the cancer cells through tumor immune recognition, cytokine release, and immune cell recruitment.

[0199] For example, the application of chimeric antigen receptors (CARs) to immunogene therapy of malignant tumors is a promising strategy in which an antibody or ligand-binding domain is fused to the zeta signaling chain of the T cell receptor. The resulting CAR immune cells are redirected with neospecificity to attack tumors expressing the surface antigen or receptor(s) recognized by the genetically engineered T cell receptor, providing a cellular therapy that attacks tumors via the normal host immune response in a highly regulated manner. These cells circulate freely in the brain and systemic circulation, eliminating the need for colocalization and bioavailability.

[0200] Multiple generations of CAR immune cells have been developed. CARs are created by fusing a tumor-specific scFv antibody or other extracellular ligand-binding domain with a TCR-associated CD3ζ signaling domain or another intracellular signaling domain from a costimulatory protein receptor. This structure allows CARs to have the tumor specificity of a B cell antigen receptor and activate T cells through the T cell antigen receptor independently of MHC binding. First-generation CARs contain one intracellular signaling domain, usually accompanied by a CD3ζ signaling domain, which enables TCR signaling. Second-generation CARs have two intracellular signaling domains: a costimulatory domain containing either a CD28 or 4-1BB signaling domain combined with a CD3ζ signaling domain. This arrangement allows T cell activation and proliferation upon antigen recognition by the scFv region of the CAR. Third-generation CARs have two intracellular signaling domains: a CD3ζ signaling domain and a costimulatory domain containing either a CD28 or 4-1BB signaling domain. This arrangement allows T cell activation and proliferation upon antigen recognition by the scFv region of the CAR. and one CD3ζ signaling domain. The first costimulatory domain is either a CD28 or 4-1BB domain, and the second costimulatory domain is either a CD28, 4-1BB, or OX40 domain. Fourth-generation "armed CAR T cells" combine second-generation CARs with the addition of various genes, including cytokines and costimulatory ligands, to enhance the tumoricidal effect of CAR T cells. See, e.g., Batlevi et al. (2016) Nature Reviews Clinical Oncology 13:25-40. See also U.S. Patent No. 7,741,465 and International Patent Publication No. WO2014 / 127261, all of which are incorporated herein by reference in their entireties.

[0201] Alternative approaches to T cell targeting include the "Trifunctional T cell antigen coupler and The TACs include the T cell antigen couplers described in International Application No. WO 2015 / 117229, entitled "Methods and Uses thereof." The T cell antigen coupler system contains three linking domains: a target-specific polypeptide ligand; a ligand that binds to a protein associated with the TCR complex, e.g., an scFv binding to CD3 (TCR, T cell receptor), which stimulates T cell activation; and a T cell receptor signaling domain, e.g., the CD4 transmembrane and intracellular domains, which amplify T cell activation. By stimulating T cell activation via the TCR, the TACs have been engineered to interface with key molecular mechanisms of T cells.

[0202] Antibody-linked T cell receptors are another approach to T cell targeting. ACTR is a hybrid approach to CARs and established monoclonal antibody tumor therapeutics. ACTR consists of a typical CAR construct that can bind to the heavy chain of an antibody via a high-affinity variant of the Fc receptor CD16. ACTR-T cells can target tumors by binding ligands that target specific cancer antigens. T cell activation is carried out by the CAR module.

[0203] Bispecific T cell exchangers (BiTEs) are bispecific antibodies that can bind to the TCR of T cells and target tumor cells via two modules: a cancer-targeting ligand; and a CD3-binding scFv domain that bridges T cells to the tumor.

[0204] Targeted therapies against IL13Rα2 have been developed, including bacterial toxins, nanoparticles, and oncolytic viruses conjugated to IL13, as well as immunotherapies using monoclonal antibodies, IL13Rα2-pulsed dendritic cells, and IL13Rα2-targeted chimeric antigen receptors (see Kahlon et al. (2004) Cancer Research. 64(24):9160-9166; Kong et al. (2012) Clinical Cancer Research. 18(21):5949-5960; Thaci et al. (2014) Neuro-Oncology; and clinical trials NCT02208362, NCT00730613, and NCT01082926). In some embodiments, these targeted therapies can be used to deliver IL-2 muteins to tumors.

[0205] The biological properties that provide for selective alteration of IL-13 activity are of interest for many therapeutic purposes, including the treatment of certain cancers through manipulation of T cell specificity, and the present invention addresses this problem.

[0206] Methods for enhancing anti-tumor immune effector cells, such as T cells, NK cells, etc., using targeted compositions including, but not limited to, chimeric antigen receptors (CARs); T cell antigen conjugates (TACs); antibody-coupled T cell receptors (ACTRs); and bispecific T cell exchangers (BiTEs) in which IL-13 or IL-4 superkines provide target-specific ligands. Methods and compositions are provided. In a further embodiment, the immune effector cells express an IL-2 mutein.

[0207] Immune cell targeting or expression constructs comprising IL-2 superkine sequences are provided and can include any of the IL-2 sequences described herein. Superkines are useful for targeting immune cells to cells expressing at least one receptor, e.g., tumor cells. 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 90% identical to any one of SEQ ID NO:2, or SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, substitutions in the IL-2 mutein include L80F, R81D, L85V, I86V, and I92F, numbered according to wild-type human IL-2 in SEQ ID NO:2.

[0208] The IL-2 superkine or mutein component of the construct can be at least about 50 amino acids in length, at least about 75, at least about 100, at least about 110, or at least about 115 amino acids in length, up to the full length of the wild-type protein in the transmembrane domain, i.e., about 116 amino acids in length. For example, the superkine or mutein can be fused to the hinge, transmembrane, or signaling domain of a CAR. Exemplary polypeptide sequences are provided.

[0209] Included as superkines or muteins are amino acid and nucleic acid coding sequences that are 90%, 95%, 98%, or 99% identical to these sequences, longer sequences that include these sequences but contain additional nucleotides at the 3' or 5' end, e.g., any number of additional nucleotides or codons, such as 3, 6, 9, 12 or more nucleotides, or up to about 12, 20, 50, or 100 additional nucleotides, and any sequences that, due to the degeneracy of the genetic code, encode the same amino acid sequence as these nucleic acids. In particular, codon-optimized (CO) sequences for expression by a desired host are contemplated as part of the invention. In some embodiments, the amino acid sequences are 90% identical. In some embodiments, the amino acid sequences are 95% identical. In some embodiments, the amino acid sequences are 98% identical. In some embodiments, the amino acid sequences are 99% identical. In some embodiments, the polypeptide is linked to an IL-2 superkine immune cell targeting or expression construct. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise one or more signaling domains derived from CD3-zeta, CD28, DAP10, OX-40, ICOS, and CD137. In some embodiments, IL-2 superkine immune cell targeting or expression constructs or expression constructs comprise one or more signaling domains derived from CD3-zeta. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise one or more signaling domains derived from CD28. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise one or more signaling domains derived from DAP10. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise one or more signaling domains derived from OX-40. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise one or more signaling domains derived from CD137. In some embodiments, IL-2 superkine immune cell targeting or expression constructs comprise IL-2 variants / IL-2 superkines, including those provided herein.In some embodiments, the IL-2 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, including those provided in SEQ ID NO:2 through SEQ ID NO:38.

[0210] 1.NK cells In some embodiments, the immune cells are natural killer (NK) cells. NK cells recognize infected or transformed cells through multiple cell surface receptors, including NKG2D, CD16, and natural cytotoxicity receptors (NCRs), such as NKp44, NKp46, and NKp30. These receptors contain immunotyrosine-based activation motifs (ITAMs), which initiate the release of cytolytic granules containing perforin and granzymes, and activate signaling adaptor proteins, such as DAP10, DAP12, and CD3ζ, which mediate the production and release of cytokines and chemokines, such as IFN-γ and TNF-α. Importantly, NK cell-mediated cytotoxicity is independent of the presentation of autologous HLA. Therefore, NK cells hold significant clinical interest as cell-based cancer therapies due to their ability to be used in allogeneic settings and potentially provide commercially available cell products.

[0211] Natural killer cells (NK cells) are an alternative to T cells for adoptive immunotherapy because they do not require HLA matching and can be used as allogeneic effector cells. Clinical trials of adoptively transferred allogeneic NK cells have shown that these cells can survive in patients for weeks to months. Furthermore, expression of CARs on NK cells allows these cells to more effectively kill solid tumors, which are often resistant to NK cell-mediated activity, compared with hematological malignancies (particularly acute myeloid leukemia), which are typically more sensitive to NK cells. CARs useful for targeting NK cells include, for example, first-generation CAR constructs containing CD3ζ as the sole signaling domain. Second- and third-generation CARs are also useful for NK cells. In some embodiments, the ectodomain of the NK cell-activating receptor NKG2D is directly linked to CD3ζ.

[0212] NK cells for modification include cell lines or peripheral blood NK cells, which can be isolated from donors by simple blood draw or, if larger numbers of cells are needed, by apheresis. Activated PB-NK cells express a broader range of activating receptors, such as CD16, NKp44, and NKp46, as well as KIRs, which play important roles in NK cell licensing. Furthermore, PB-NK cells can be administered without irradiation and therefore have the ability to expand in vivo. Another source of NK cells suitable for CAR expression is NK cells derived from human pluripotent stem cells (both induced pluripotent stem cells (iPSCs) or human embryonic stem cells (hESCs)). These NK cells exhibit a similar phenotype to PB-NK cells, and hESC / iPSC-NK cells can be expanded on a clinical scale.

[0213] 2. Chimeric antigen receptor (CAR) In addition to the superkine sequence, CARs contain a signaling domain for CD3ζ and one or more costimulatory receptor signaling domains that further promote the recycling, survival, and / or expansion of CAR-expressing immune cells. The costimulatory receptor signaling domain is the intracellular portion of each receptor protein that generates an activation signal within the cell. Examples include amino acids 180-220 of the native CD28 molecule and amino acids 214-255 of the native 4-1BB molecule.

[0214] Examples of suitable hinge and transmembrane regions for linking the superkine to the signaling domain include, but are not limited to, the constant (Fc) region of immunoglobulin, human CD8a, and artificial linkers that serve to distance the targeting site from the cell surface to improve access and binding to the target cell. Examples of suitable transmembrane domains include the transmembrane domains of leukocyte CD markers, preferably CD4 or CD28. Examples of intracellular receptor signaling domains include the zeta chain of the T cell antigen receptor complex, preferably CD3, although any transmembrane region sufficient to anchor the CAR in the membrane can be used. Those skilled in the art are aware of numerous transmembrane regions and structural elements (such as lipophilic amino acid regions) that form transmembrane domains in many membrane proteins, and therefore can be substituted with any convenient sequence. Suitable T cell costimulatory signaling receptors for improving the function and activity of CAR-expressing cells include, but are not limited to, CD28, CD137, and OX-40.

[0215] Signaling through CD28 is necessary for IL2 production and proliferation but does not play a major role in maintaining T cell function and activity. CD137 (a member of the tumor necrosis factor receptor family that is expressed after CD28 activation) and OX-40 are involved in driving long-term T cell survival and T cell accumulation. The ligands for these receptors are usually expressed on professional antigen-presenting cells such as dendritic cells and activated macrophages, but not on tumor cells. CD4 + Expression of CARs incorporating CD28 and / or 4-1BB signaling domains in T cells enhances the activity and antitumor efficacy of these cells compared to those expressing CARs containing only the CD3ζ signaling domain, and this construct can be referred to as a second- or third-generation CAR.

[0216] CAR constructs of interest include tandem CARs, see, e.g., Hegde et al. (2016) J. ClinInvest 126(8):3036-3052, which is specifically incorporated herein by reference. In such constructs, a binding moiety of a tumor-specific antigen is tandemly combined with the IL-13 superkine. The binding moiety may be, for example, an scFv specific for a tumor cell antigen, including, but not limited to, HER-2, EGFR, CD20, etc., as known in the art.

[0217] In various embodiments, the antigen-binding domain binds to an antigen on a target cell, such as a cancer cell. The antigen-binding domain can bind to an antigen, such as, but not limited to, a tumor-targeted antigen. In some cases, the antigen-binding domain binds to one or more antigens.Exemplary antigen binding domains include CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also known as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3; TNF receptor family B-cell maturation (BCMA); Tn antigen (Tn Ag) or (GalNAcα Ser / Thr); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Rα2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); protease serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); C D20; folate receptor alpha; receptor tyrosine protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); proteasome (prosome, macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2. (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta; tumor endothelial marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor Body class C group 5 member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); phosphorylation lymphocyte antigen 6 complex, locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1); ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Ti e2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate cancer tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MART1); rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-IAP);ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired-box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytoma viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or imprinted Regulator of thyroid cancer site, squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchor protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end point (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPVE6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0218] In some embodiments, the antigen-binding domain comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a nanobody, a single-chain variable fragment (scFv), F(ab'), Fab', Fab, Fv, etc. In some embodiments, the nucleic acid encoding the antigen-binding domain is operably linked to the nucleic acid encoding the transmembrane domain of the CAR.

[0219] In some embodiments, the transmembrane domain can be derived from a membrane-bound or transmembrane protein. In certain embodiments, the transmembrane domain comprises one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more amino acid modifications (e.g., substitutions, insertions, and deletions) compared to the wild-type amino acid sequence of the transmembrane domain of the membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains of CARs include at least the transmembrane region(s) of the alpha, beta, or zeta chain of a T cell receptor, CD28, CD3 epsilon (CD3ξ), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or the erythropoietin receptor. In some embodiments, the transmembrane domain comprises a human immunoglobulin (Ig) hinge region, e.g., an IgG4 Fc hinge. In other embodiments, the transmembrane domain is a recombinant or synthetic domain that includes hydrophobic amino acid residues (e.g., leucine and valine). In some cases, the transmembrane domain includes phenylalanine, tryptophan, and valine at one or both ends of the domain.

[0220] The transmembrane domain links the antigen binding domain to the intracellular signaling domain of the CAR. In some embodiments, the nucleic acid encoding the antigen binding domain is operably linked to a nucleic acid encoding a transmembrane domain, which is operably linked to a nucleic acid encoding the intracellular signaling domain.

[0221] In some embodiments, the intracellular signaling domain of the CAR comprises a signal activation or signal transduction domain. As such, the intracellular signaling domain comprises any portion of the intracellular signaling domain of a protein sufficient to transduce or transmit a signal, e.g., an activation signal, or mediate a cellular response within the cell. Non-limiting examples include TCR, CD2, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD7, CD27, CD86, common FcR gamma, FcR beta, CD79a, CD79b, Fc gamma RIIa, DAP10, DAP12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2 , CD7, LIGHT, NKG2C, B7-H3, CD83 specific binding ligand, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD160, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD1 8, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, Ly9 (CD 229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, or any derivative, variant, or fragment thereof.In certain embodiments, the intracellular signaling domain comprises the intracellular domain of a costimulatory molecule from CD3, CD27, CD28, CD127, ICOS, 4-1BB (CD137), PD-1, a T cell receptor (TCR), any derivative thereof, or any variant thereof, etc. In some embodiments, the intracellular signaling domain of the CAR is an intracellular domain of an MHC class I molecule, a TNF receptor, or the like. Proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICO S(CD278), GITR, BAFFR, LIGHT, HVEM(LIGHTR), KIRDS2, SLAMF7, NKp80(KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGA D, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITG B7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRTAM, L The ligand is selected from the group consisting of ligands that specifically bind to y9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0222] 3.BiTES Bi-specific T-cell engagers (BiTEs) are fusion proteins comprising an IL-13 superkine fused to an antibody variable region that specifically binds to CD3. In some embodiments, the antibody variable region is a single-chain variable fragment (scFv). The superkine may be fused to the variable region via a linker. An Fc region is optionally provided.

[0223] 4.TAC The TAC construct comprises an IL-2 superkine fused to a ligand that binds to a protein associated with the TCR complex and fused to a T cell receptor signaling domain polypeptide. The domains may be separated by a linker. The protein associated with the TCR complex may be CD3. The ligand that binds to a protein associated with the TCR complex may be a single-chain antibody. The ligand that binds to a protein associated with the TCR complex may be UCHT1 or a variant thereof. The T cell receptor signaling domain polypeptide may include a cytosolic domain and a transmembrane domain. The cytosolic domain may be a CD4 cytosolic domain, and the transmembrane domain may be a CD4 transmembrane domain.

[0224] 5.ACTR ACTR is a hybrid approach to CARs and established monoclonal antibody tumor therapeutics. ACTR consists of a typical CAR construct that can bind to the heavy chain of an antibody via a high-affinity variant of the Fc receptor CD16. A superkine is fused to the moiety recognized by the CAR, including but not limited to the Fc region of an antibody with high affinity for CD16.

[0225] The coding sequence of the immune cell targeting or expression construct can be generated by any means known in the art, including recombinant DNA technology. Nucleic acids encoding several regions of the chimeric receptor can be conveniently prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (e.g., genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.). The resulting coding region can be inserted into an expression vector and expressed in an appropriate expression host cell line, e.g., primary culture medium. This method can be used to transform populations of allogeneic or autologous T lymphocytes, allogeneic or autologous NK cells, including cultures, cell lines, iPSC-derived cells, etc. This method can be used in vitro (e.g., cell-free systems), in culture, e.g., in vitro or ex vivo, on cells. For example, IL-2 superkine CAR-expressing cells can be cultured and expanded in vitro in culture medium.

[0226] Non-IL-2 superkine immune cell targeting or expression constructs can be used to specifically direct immune cells to target specific tumor cells. + or CD8 + Anti-tumor effector cells, such as effector T cells, can be generated by introducing superkine immune cell targeting or expression constructs containing one or more signaling domains from CD3ζ, CD28, DAP10, OX-40, ICOS, or CD137 into T cells so that they are redirected to recognize such tumor cells. In some embodiments, the cells can further contain a transgene capable of expressing an IL-2 mutein described herein. IL-2 superkine immune cell targeting or expression constructs can specifically direct immune cells to target IL-2R-expressing cells, including tumor cells. CD4 + or CD8 +Anti-tumor effector cells, such as effector T cells, can be generated and redirected to recognize such tumor cells by introducing into T cells IL-2 superkine immune cell targeting or expression constructs containing one or more signaling domains derived from CD3ζ, CD28, DAP10, OX-40, ICOS, and CD137.

[0227] The IL-2 superkine immune cell targeting or expression construct is transfected or transfected into human immune cells using, for example, non-viral plasmid vectors and electroporation methods, viral vectors and infection methods, etc., as known in the art. CARs containing costimulatory signaling domains can enhance the duration and / or retention of antitumor activity, which can significantly improve the clinical efficacy of adoptive therapy protocols. CD4 + and CD8 + T cell effector function, as well as NK cell function, can be driven via these receptors, and therefore these cell types are contemplated for use in the present invention. + T cells can be used to lyse target cells and produce IL-2 in the presence of target cells, among other functions of these cells. + and CD8 + Expression of an appropriate costimulatory CAR on either or both of the T cells is used for adoptive immunotherapy, thus providing the most effective cell population, consisting of either or both professional helper and killer T cells, that exhibit enhanced and / or prolonged survival and anti-tumor activity. In some embodiments, the IL-2 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, including those provided in Figure 2. In some embodiments, the IL-2 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, including any provided herein.

[0228] The polypeptides of the present invention can be further modified, e.g., conjugated to a wide variety of other oligopeptides or proteins for various purposes. For example, they can be post-translationally modified by prenylation, acetylation, amidation, carboxylation, glycosylation, pegylation, etc. Such modifications can also include glycosylation modifications, e.g., made by modifying the glycosylation pattern of the polypeptide during its synthesis and processing or further processing steps, e.g., made by exposing the polypeptide to enzymes that affect glycosylation, such as mammalian glycosylation or deglycosylation enzymes.

[0229] Methods well known to those skilled in the art can be used to construct T cell targeting construct expression vectors containing the coding sequence and appropriate transcriptional / translational control signals. These methods include, for example, These include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombination / genetic recombination. Alternatively, RNA capable of encoding a polypeptide of interest may be chemically synthesized. Those skilled in the art can readily utilize well-known codon usage tables and synthetic methods to provide suitable coding sequences for any of the polypeptides of the present invention. Nucleic acids may be isolated and obtained with substantial purity. Typically, nucleic acids, either DNA or RNA, may be obtained substantially free of other naturally occurring nucleic acid sequences, generally at least about 50%, usually at least about 90% pure, and typically in "recombinant" form, e.g., flanked by one or more nucleotides not normally associated on the chromosome in which they occur in nature. Nucleic acids of the present invention can be provided as linear or circular molecules, and can be provided in autonomously replicating molecules (vectors) or molecules lacking replication sequences. Expression of the nucleic acid may be regulated by its own or other regulatory sequences known in the art. Nucleic acids of the present invention can be introduced into suitable host cells using a variety of techniques available in the art.

[0230] In accordance with the present invention, immune cell targeting or expression construct vectors and immune cell targeting or expression construct-modified cells can be provided in pharmaceutical compositions suitable for therapeutic use, e.g., human treatment. In some embodiments, pharmaceutical compositions of the present invention comprise one or more therapeutic agents of the present invention, or pharmaceutically acceptable salts, esters, or solvates thereof. In some other embodiments, pharmaceutical compositions of the present invention comprise one or more therapeutic agents of the present invention in combination with another therapeutic agent, e.g., another anti-tumor agent.

[0231] Therapeutic agents of the present invention are often administered as pharmaceutical compositions containing an active therapeutic agent and another pharmaceutically acceptable excipient. Such formulations may contain one or more non-toxic pharmaceutically acceptable carriers, diluents, excipients, and / or adjuvants. The preferred form depends on the intended mode of administration and therapeutic use. Depending on the desired formulation, the composition may also contain a pharmaceutically acceptable non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc.

[0232] In still other embodiments, the pharmaceutical compositions of the present invention comprise large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized sepharose). TM , agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).

[0233] The maximum tolerated dose (MTD) of the CAR immune cells may be determined during clinical trial development, for example, up to about 10 as empirically determined. 4 T cells / kg body weight, up to about 105 cells / kg body weight, up to about 10 6 cells / kg body weight, up to approximately 5×10 6 cells / kg body weight, up to about 10 7 cells / kg body weight, up to approximately 5×10 7 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 cells / kg body weight or more. 4 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 5 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 6 In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 10 7 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5 x 10 6 T cells / kg body weight. In some embodiments, the maximum tolerated dose (MTD) of CAR immune cells is up to about 5 x 10 7 T cells / kg body weight.

[0234] The toxicity of the cells described herein can be determined by standard pharmaceutical techniques in cell culture or experimental animals, e.g., LD 50 (Lethal dose for 50% of the population) or LD 100 The therapeutic index can be determined by determining the lethal dose (100% lethal dose in a population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Data obtained from these cell culture assays and animal studies can be used to formulate a non-toxic dosage range for human use. The dosages described herein preferably fall within a range of circulating concentrations that include the effective dose with little or no toxicity. Dosages can vary within this range depending on the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition.

[0235] After the dose escalation study, patients in the expansion cohort are treated with immune cells at the MTD. Exemplary treatment regimens involve administration once every two weeks, once a month, or once every three to six months. The therapeutic agents of the present invention are typically administered on multiple occasions. The intervals between single doses can be weekly, monthly, or yearly. The intervals can be irregular, as indicated by measuring the blood concentration of the therapeutic agent in the patient.

[0236] In prophylactic use, for example, a relatively low dosage can be administered at relatively infrequent intervals for a long period of time to maintain the patient's remission.Some patients continue to receive treatment for the rest of their lives.In other therapeutic use, a relatively high dosage may be required at relatively short intervals until the progression of the disease is reduced or terminated, preferably until the patient shows partial or complete improvement of the symptoms of the disease.Then, the patient can be administered a prophylactic regimen.

[0237] Examples of additional therapeutic agents that can be administered and / or formulated with immune cell targeting or expression constructs include antiproliferative or cytoreductive therapies, which are used therapeutically to eliminate tumor cells and other unwanted cells in a host and include the use of treatments such as the delivery of ionizing radiation and the administration of chemotherapeutic agents. Chemotherapeutic agents are well known in the art and are used at conventional or reduced doses and regimens, and include topoisomerase inhibitors such as anthracyclines, including the compounds daunorubicin, adriamycin (doxorubicin), epirubicin, idarubicin, annamycin, and MEN10755. Other topoisomerase inhibitors include the podophyllotoxin analogs etoposide and teniposide, as well as anthracenedione, mitoxantrone, and amsacrine. Other antiproliferative agents interfere with microtubule assembly, such as the vinca alkaloid family. Examples of vinca alkaloids include vinblastine, vincristine, vinorelbine (NAVELBINE), vindesine, vindoline, vincamine, etc. DNA damaging agents include nucleotide analogs, alkylating agents, etc. Alkylating agents include nitrogen mustards, such as mechlorethamine, cyclophosphamide, melphalan (L-sarcolysin), etc., and nitro sources, such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin, etc. Nucleotide analogs include pyrimidines, such as cytarabine (CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), and floxuridine (FUdR); purines, such as thioguanine (6-thioguanine), mercaptopurine (6-MP), pentostatin, and fluorouracil (5-FU); and folic acid analogs, such as methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717), 5,8-dideazatetrahydrofolic acid (DDATHF), and leucovorin. Other chemotherapeutic agents of interest include metal complexes, such as cisplatin (cis-DDP), carboplatin, and oxaliplatin; ureas, such as hydroxyurea, and hydrazines, such as N-methylhydrazine.

[0238] For example, ionizing radiation (IR) is used to treat approximately 60% of cancer patients by depositing energy that damages or destroys cells in the treated area. For purposes of this invention, it can be delivered at conventional doses and regimens, or at lower doses. Radiation damage to cells is nonspecific and has complex effects on DNA. The effectiveness of treatment depends on greater cytotoxicity in cancer cells than in normal cells. Radiation therapy can be used to treat all types of cancer. Some types of radiation therapy involve photons, such as X-rays and gamma rays. Another technique for delivering radiation to cancer cells is internal radiation therapy, which focuses the radiation dose into a small area by placing a radioactive implant directly into the tumor or body cavity. Suitable doses of ionizing radiation can range from at least about 2 Gy to no more than about 10 Gy, usually about 5 Gy. Suitable doses of ultraviolet radiation are at least about 5 J / m. 2 ~about 50J / m 2 Range below 10J / m, typically around 10J / m 2 The sample may be collected within at least about 4 hours to about 72 hours, usually about 4 hours, after UV irradiation.

[0239] The treatment may be combined with immunomodulatory agents, including (iii) agents that stimulate immune costimulatory molecules, such as CD40 and OX40, and / or (iv) agents that antagonize immune suppressive molecules, such as CTLA-4, PD-1, and PD-L1. The active agents are administered within a period of time to produce additive or synergistic effects on the depletion of cancer cells in the host. Administration methods include, but are not limited to, systemic administration, intratumoral administration, etc.

[0240] In some embodiments, the cancer is selected for treatment with the combination therapy because the cancer is a cancer type that responds to checkpoint inhibitors, such as PD-1 antagonists, PD-L1 antagonists, CTLA4 antagonists, TIM-3 antagonists, BTLA antagonists, VISTA antagonists, LAG3 antagonists, etc. In some embodiments, the immunomodulatory agent is a CTLA-4, PD1, or PDL1 antagonist, such as avelumab, nivolumab, pembrolizumab, ipilimumab, etc. In some such embodiments, the cancer is, but is not limited to, melanoma or small cell lung cancer. In some such embodiments, the cancer is a type with a high neoantigen or mutagenesis burden (see Vogelstein et al. (2013) Science 339(6127):1546-1558, specifically incorporated herein by reference).

[0241] In some embodiments, the cancer is selected for treatment with the combination therapy of the present invention because the cancer is a cancer type that responds to immune response agonists, such as CD28 agonists, OX40 agonists, GITR agonists, CD137 agonists, CD27 agonists, HVEM agonists, etc. In some embodiments, such immunomodulatory agents are OX40, CD137, or GITR agonists, such as tremelimumab. In some such embodiments, the cancer is, but is not limited to, melanoma or small cell lung cancer. In some such embodiments, the cancer is a type with high neoantigen or mutation burden.

[0242] In some embodiments, the combination therapy includes an antibody known in the art that binds to PD-1 and disrupts the interaction between PD-1 and its ligand PD-L1, stimulating an anti-tumor immune response. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. For example, antibodies that target PD-1 and can be used in the present invention include, for example, nivolumab (BMS-936558, Bristol-Myers Squibb), pembrolizumab (lambrolizumab, MK03475 or MK-3475, Merck), humanized anti-PD-1 antibody JS001 (ShangHai JunShi), monoclonal anti-PD-1 antibody TSR-042 (Tesaro, Inc.), pidilizumab (anti-PD-1 mAb CT-011, Medivation), anti-PD-1 monoclonal antibody BGB-A317 (BeiGene), and / or anti-PD-1 antibody SHR-12. Suitable antibodies include, but are not limited to, PD-1 antibodies derived from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Suitable antibodies include, but are not limited to, PD-L1 10 (ShangHai HengRui), human monoclonal antibody REGN2810 (Regeneron), human monoclonal antibody MDX-1106 (Bristol-Myers Squibb), and / or humanized anti-PD-1 IgG4 antibody PDR001 (Novartis). In some embodiments, the PD-1 antibody is derived from clone: ​​RMP1-14 (rat IgG) - BioXcell catalog number BP0146. Other suitable antibodies include the anti-PD-1 antibodies disclosed in U.S. Patent No. 8,008,449, incorporated herein by reference. In some embodiments, the antibody or antigen-binding portion thereof specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity. Any antibody known in the art that binds to PD-L1, disrupts the interaction between PD-1 and PD-L1, and stimulates an anti-tumor immune response is suitable for use in the combination therapies disclosed herein. For example, antibodies targeting PD-L1 that are in clinical trials include BMS-936559 (Bristol-Myers Squibb) and MPDL3280A (Genetech). Other suitable antibodies that target PD-L1 are disclosed in U.S. Patent No. 7,943,743, which is incorporated herein by reference. Those skilled in the art will appreciate that any antibody that binds to PD-1 or PD-L1, disrupts the PD-1 / PD-L1 interaction, and stimulates an anti-tumor immune response is suitable for use in the combination therapies disclosed herein.

[0243] In some embodiments, the combination therapy includes an antibody known in the art that binds to CTLA-4 and disrupts its interaction with CD80 and CD86. Exemplary antibodies that target CTLA-4 include FDA-approved ipilimumab (MDX-010, MDX-101, Bristol-Myers Squibb) and tremelimumab (ticilimumab, CP-675, 206, Pfizer), which is currently undergoing human trials. Other suitable antibodies that target CTLA-4 are disclosed in WO 2012 / 120125, U.S. Patent Nos. 6,984,720 and 6,682,7368, and U.S. Patent Application Nos. 2002 / 0039581, 2002 / 0086014, and 2005 / 0201994, which are incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to CTLA-4, disrupts its interaction with CD80 and CD86, and stimulates an anti-tumor immune response is suitable for use in the combination therapy disclosed herein. In some embodiments, the combination therapy includes an antibody known in the art that binds to LAG-3 and disrupts its interaction with MHC class II molecules. An exemplary antibody that targets LAG-3 is IMP321 (Immutep), which is currently in human trials. Other suitable antibodies that target LAG-3 are disclosed in U.S. Patent Application No. 2011 / 0150892, which is incorporated herein by reference. Those skilled in the art will understand that any antibody that binds to LAG-3, disrupts its interaction with MHC class II molecules, and stimulates an anti-tumor immune response is suitable for use in the combination therapy disclosed herein.

[0244] In some embodiments, the combination therapy includes an antibody known in the art that binds to TIM-3 and disrupts its interaction with Galectin 9. Suitable antibodies that target TIM-3 are disclosed in U.S. Patent Application Publication No. 2013 / 0022623, which is incorporated herein by reference. One of skill in the art will appreciate that any antibody that binds to TIM-3, disrupts its interaction with Galectin 9, and stimulates an anti-tumor immune response is suitable for use in the combination therapy methods disclosed herein.

[0245] In some embodiments, the combination therapy includes an antibody known in the art that binds to 4-1BB / CD137 and disrupts its interaction with CD137L. One of skill in the art will understand that antibodies that bind to 4-1BB / CD137, disrupt its interaction with CD137L or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0246] In some embodiments, the combination therapy includes an antibody known in the art that binds to GITR and disrupts its interaction with its ligand. It will be understood by those skilled in the art that antibodies that bind to GITR, disrupt its interaction with GITRL or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy disclosed herein.

[0247] In some embodiments, the combination therapy includes an antibody known in the art that binds to OX40 and disrupts its interaction with its ligand. One of skill in the art will understand that antibodies that bind to OX40, disrupt its interaction with OX40L or another ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0248] In some embodiments, the combination therapy includes antibodies known in the art that bind to CD40 and disrupt its interaction with its ligand. Those skilled in the art will understand that antibodies that bind to CD40, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0249] In some embodiments, the combination therapy includes an antibody known in the art that binds to ICOS and disrupts its interaction with its ligand. Those skilled in the art will understand that antibodies that bind to ICOS, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0250] In some embodiments, the combination therapy includes antibodies known in the art that bind to CD28 and disrupt its interaction with its ligand. Those skilled in the art will understand that antibodies that bind to CD28, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0251] In some embodiments, the combination therapy includes antibodies known in the art that bind to IFNα and disrupt its interaction with its ligand. Those skilled in the art will understand that antibodies that bind to IFNα, disrupt its interaction with its ligand, and stimulate an anti-tumor immune response or immunostimulatory response that collectively results in anti-tumor activity are suitable for use in the combination therapy methods disclosed herein.

[0252] An "anti-cancer therapeutic agent" is a compound, composition, or treatment (such as surgery) that prevents or delays the growth and / or metastasis of cancer cells. Such anti-cancer treatments include, but are not limited to, surgery (e.g., removal of all or part of a tumor), chemotherapy treatments, radiation, gene therapy, hormone manipulation, immunotherapy (e.g., therapeutic antibodies and cancer vaccines), and antisense or RNAi oligonucleotide therapy. Examples of useful chemotherapeutic agents include hydroxyurea, busulfan, cisplatin, carboplatin, chlorambucil, melphalan, cyclophosphamide, ifosfamide, danorubicin, doxorubicin, epirubicin, mitoxin, riboflavin ... These include, but are not limited to, santrone, vincristine, vinblastine, navelbine (vinorelbine), etoposide, teniposide, paclitaxel, docetaxel, gemcitabine, cytosine arabinoside, bleomycin, neocarcinostatin, suramin, taxol, mitomycin C, avastin, herceptin (herceptin), fluorouracil, and temozolamide. The compounds are also suitable for use in standard combination therapy using two or more chemotherapeutic agents. Anticancer treatments include the development of new anticancer drugs. It is to be understood that the present invention includes novel compounds or treatments that are being investigated.

[0253] The above-mentioned pharmaceutical compositions and / or formulations contain one or more therapeutic agents in an amount effective to achieve the intended purpose. Thus, the term "therapeutically effective amount" refers to the amount of a therapeutic agent that improves the symptoms of cancer. Determining the therapeutically effective dose of a compound is well within the capabilities of those skilled in the art. For example, the therapeutically effective amount can be initially estimated using either cell culture assays or animal models as described herein. Animal models can also be used to determine appropriate concentration ranges and administration routes. Such information can then be used to determine useful doses and routes for administration in other animals, including humans, using standard methods known to those skilled in the art.

[0254] A kit comprising the composition of the present invention and instructions for use is also within the scope of the present invention. The kit may further comprise at least one additional reagent, such as a chemotherapeutic agent, an anti-tumor antibody, etc. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material on or accompanying the kit, or otherwise associated with the kit.

[0255]

[0043] Having fully described the invention, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. In some embodiments, a kit comprises an IL-2 superkine immune cell targeting or expression construct comprising an IL-2 variant / IL-2 superkine described herein. In some embodiments, a kit comprises an IL-2 superkine immune cell targeting or expression construct comprising an IL-2 variant / IL-2 superkine, including those provided herein. In some embodiments, an IL-2 superkine immune cell targeting or expression construct comprises an IL-2 variant / IL-2 superkine, including those provided herein.

[0256] 6. Exemplary Immune Cell Targeting or Expression Construct Embodiments The immune cell targeting or expression construct comprises an interleukin-2 receptor beta (IL-2Rβ) binding protein, wherein the equilibrium dissociation constant of said binding protein with IL-2Rβ is less than that of wild-type human IL-2 (hIL-2); linked to the immune cell targeting or expression construct.

[0257] In some embodiments, the immune cell targeting or expression construct exhibits a cytotoxic effect on T cells, e.g., CD8+ T cells or CD4+ T cells.

[0258] In some embodiments, the construct is a chimeric antigen receptor (CAR), in which the IL-2 superkine is fused to a transmembrane domain and linked to an intracellular signaling region.

[0259] In some embodiments, the intracellular signaling region comprises a CD3 signaling domain.

[0260] In some embodiments, the intracellular signaling region comprises one or more of a CD28 signaling domain, a CD137 signaling domain, an OX-40 signaling domain, an ICOS signaling domain, a DAP10 signaling domain.

[0261] In some embodiments, the construct is a T cell antigen conjugate (TAC), in which the IL-2 superkine is fused to a ligand that binds to a protein associated with the TCR complex; and to a T cell receptor signaling domain polypeptide.

[0262] In some embodiments, the protein associated with the TCR complex is CD3.

[0263] In some embodiments, the T cell receptor signaling domain polypeptide comprises a CD4 cytosolic domain and a CD4 transmembrane domain.

[0264] In some embodiments, the construct is an antibody-binding T-cell receptor (ACTR) that comprises a chimeric antigen receptor component that binds with high affinity to the IL-2 superkine.

[0265] In some embodiments, the CAR component comprises CD16 and the IL-2 superkine is fused to an Fc sequence.

[0266] In some embodiments, the construct is a bispecific T cell exchanger (BiTE) comprising an IL-2 superkine fused to the variable region of an antibody that binds a component of the T cell receptor.

[0267] In some embodiments, the BiTE component of the T cell receptor is CD3.

[0268] In some embodiments, the IL-2Rβ binding protein comprises the following amino acid substitutions, numbered according to wild-type hIL-2: L80F, R81D, L85V, I86V, and I92F.

[0269] In some embodiments, a nucleic acid described herein and encoding IL-2 is provided. In some embodiments, a vector comprising the nucleic acid is provided.

[0270] In some embodiments, a T cell is provided comprising a construct according to any of the above. In some embodiments, a NK cell is provided comprising a construct according to any of the above. In some aspects, the T cell is CD4 + In some embodiments, the T cells are CD8 + T cells.

[0271] Also provided are isolated populations of the above immune cells. Pharmaceutical formulations comprising the above immune cell populations are also provided.

[0272] H. Expression of mutant IL-2 gene products The nucleic acid molecules described above can be contained within, for example, vectors that are capable of directing their expression in cells transduced with the vector. Thus, in addition to the subject IL-2 muteins, expression vectors containing nucleic acid molecules encoding the subject IL-2 muteins and cells transfected with these vectors are included in preferred embodiments.

[0273] It should be understood, of course, that not all vectors and expression control sequences will function equally well to express the DNA sequences described herein. Also, not all hosts will function equally well in the same expression system. However, one of ordinary skill in the art can select 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 in the host. The vector's copy number, the ability to control that copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, must also be considered. For example, vectors that can be used include those that can amplify the copy number of DNA encoding an IL-2 mutein. Such amplifiable vectors are well known in the art. They include, for example, DHFR amplification (e.g., Kaufman, U.S. Pat. No. 4,470,461; Kaufman and Sharp, "Construction of a Modular Dihydrafolate Reductase cDNA Gene: Analysis of Signals Utilized"). for Efficient Expression”, Mol.Cell.Biol ., 2, pp. 1304-19 (1982)) or glutamine synthetase ("GS") amplification (see, e.g., U.S. Pat. No. 5,122,464 and European Published Application No. 338,841).

[0274] In some embodiments, the human IL-2 muteins of the present disclosure will be expressed from a vector, preferably an expression vector. Vectors can be useful for autonomous replication in host cells or can be integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome (e.g., non-episomal mammalian vectors). Expression vectors are capable of directing the expression of coding sequences to which they are operably linked. 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.

[0275] Exemplary recombinant expression vectors are selected based on the host cell to be used for expression and can include one or more regulatory sequences operably linked to the nucleic acid sequence to be expressed.

[0276] Expression constructs or vectors can be designed for expression of an IL-2 mutein or variant thereof in prokaryotic or eukaryotic host cells.

[0277] 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.

[0278] Protein expression in prokaryotes is most often carried out in Escherichia coli using vectors containing constitutive or inducible promoters. Strategies for maximizing recombinant protein expression in E. coli can be found, for example, in Gottesman (1990) Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.), pp. 119-128 and Wada et al. (1992) Nucleic Acids Res. 20: 2111-2118. Processes for growing, harvesting, disrupting, or extracting IL-2 muteins or variants thereof from cells are substantially as described, for example, in U.S. Pat. Nos. 4,604,377; 4,738,927; 4,656,132; 4,569,790; 4,748,234; 4,530,787; 4,572,798; 4,748,234; and 4,931,543, which are incorporated herein by reference in their entireties.

[0279] In some embodiments, recombinant IL-2 muteins or biologically active variants thereof can also be produced in eukaryotes, such as yeast or human cells. Suitable eukaryotic host cells include insect cells (examples of baculovirus vectors available for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39)); yeast cells (examples of vectors for expression in the yeast S. cerenvisiae include pYepSec1 (Baldari et al. (1987) EMBOJ. 6:229-234), pMFa (Kurjan and Herskowitz (1982) Cell 30:9)); 33-943), pJRY88 (Schultz et al. (1987) Gene 54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and pPicZ (Invitrogen Corporation, San Diego, Calif.); or mammalian cells (mammalian expression vectors include pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187:195)). Suitable mammalian cells include Chinese hamster ovary cells (CHO) or COS cells. In mammalian cells, the control functions of the expression vector are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. For other expression systems suitable for both prokaryotic and eukaryotic cells, see Chapters 16 and 17, Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2 nd ed., Cold Spring Harbor Laboratory Press, Plainview, NY. See Goeddel (1990) Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, Calif.).

[0280] The sequences encoding the human IL-2 muteins of the present disclosure can be optimized for expression in a target host cell. The GC content of the sequence can be adjusted to an average level for a particular cellular host, as calculated with reference to known genes expressed in the host cell. Methods for codon optimization are well known in the art. The codons in the IL-2 mutein coding sequence can be optimized to enhance expression in the host cell, resulting in about 1%, about 5%, about 10%, about 25%, about 50%, about 75%, or up to 100% of the codons in the coding sequence being optimized for expression in a particular host cell.

[0281] Vectors suitable for use include T7-based vectors for bacteria (see, e.g., Rosenberg et al., Gene 56:125, 1987), the pMSXND expression vector for mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), and baculovirus-derived vectors for insect cells (e.g., the expression vector pBacPAK9 from Clontech, Palo Alto, Calif.).

[0282] In some embodiments, the nucleic acid insert encoding the subject IL-2 mutein in such a vector can be operably linked to a promoter selected, for example, based on the cell type in which expression is desired.

[0283] Various factors must also be considered when selecting an expression control sequence. These include, for example, the relative strength of the sequence, its controllability, particularly with respect to potential secondary structure, and compatibility with the actual DNA sequence encoding the IL-2 mutein of interest. A host should be selected taking into consideration compatibility with the selected vector, toxicity of the product encoded by the DNA sequence of the invention, secretion characteristics, ability to properly fold the polypeptide, fermentation or cultivation requirements, and ease of purification of the product encoded by the DNA sequence.

[0284] Within these parameters, one skilled in the art can select a variety of vector / expression control sequence / host combinations that will express the desired DNA sequence in fermentation or large scale animal culture using, for example, CHO or COS7 cells.

[0285] In some embodiments, the choice of expression control sequence and expression vector depends on the choice of host. A wide variety of expression host / vector combinations can be used. Useful expression vectors for eukaryotic hosts include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include bacterial plasmids such as E. coli-derived plasmids, including colEl, pCRI, pER32z, pMB9, and their derivatives, broader host range plasmids, e.g., RP4, phage DNA, e.g., many derivatives of phage lambda, e.g., NM989, and other DNA phages, e.g., M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells include the 2μ plasmid and its derivatives. Vectors useful for insect cells include pVL941 and pFastBac™1 (GibcoBRL, Gaithersburg, Md.). Cate et al., "Isolation of the Bovine and Human Genes for Mullerian Inhibiting Substance and Expression of the Human Gene in Animal Cells," Cell, 45, pp. 685-98 (1986).

[0286] Furthermore, any of a 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 aforementioned expression vectors. Examples of useful expression control sequences include, for example, the early and late promoters of SV40 or adenovirus, the lac system, the trp system, the TAC or TRC system, the major operator and promoter regions of phage lambda, e.g., PL, the fd coat protein control region, promoters of 3-phosphoglycerate kinase or other glycolytic enzymes, acid phosphatase promoters, e.g., PhoA, yeast α-mating system promoters, baculovirus polyhedron promoters, and other sequences known to control the expression of genes in prokaryotic or eukaryotic cells or their viruses, as well as various combinations thereof.

[0287] The T7 promoter can be used in bacteria, the polyhedrin promoter can be used in insect cells, and the cytomegalovirus or metallothionein promoter can be used in mammalian cells. In addition, in the case of higher eukaryotes, tissue-specific and cell type-specific promoters are widely available. These promoters are so named because of their ability to direct the expression of nucleic acid molecules in specific tissues or cell types in the body. Those skilled in the art will be familiar with many promoters and other regulatory elements that can be used to direct the expression of nucleic acids.

[0288] In addition to sequences that facilitate transcription of the inserted nucleic acid molecule, vectors can contain an origin of replication and other genes that encode selectable markers, such as neomycin resistance (neomycin resistance). r The ) gene confers G418 resistance to cells in which it is expressed, allowing 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 situation.

[0289] Viral vectors that can be used in the present invention include, for example, retroviruses, adenoviruses and adeno-associated vectors, herpes viruses, simian virus 40 (SV40), and bovine papillomavirus vectors (see, e.g., Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, NY).

[0290] Prokaryotic or eukaryotic cells that contain and express nucleic acid molecules encoding the subject IL-2 muteins disclosed herein are also a feature of the invention. 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. Progeny of such cells also qualify as the subject IL-2 muteins of the invention. is considered to be within range.

[0291] 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 in eukaryotic hosts such as insect cells (e.g., Sf21 cells) or mammalian cells (e.g., CHO, HEK293, COS cells, NIH 3T3 cells, or HeLa cells). These cells are available from many sources, including the American Type Culture Collection (Manassas, Va.). The only important factor in selecting an expression system is that the components are compatible with each other. A specialist or skilled artisan can make such a determination. Furthermore, if guidance is needed in selecting an expression system, skilled artisans may consult Ausubel et al. (Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY, 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual, 1985 Suppl. 1987).

[0292] The expressed polypeptides can be purified from the expression system using conventional biochemical procedures and used, for example, as therapeutic agents as described herein.

[0293] In some embodiments, the resulting IL-2 mutein is glycosylated or non-glycosylated depending on the host organism used to produce the mutein. If bacteria are selected as the host, the IL-2 mutein produced will not be glycosylated. On the other hand, eukaryotic cells will glycosylate the IL-2 mutein, but perhaps not in the same manner as native IL-2 is glycosylated. The IL-2 mutein produced by the transformed host can be purified according to any suitable method. Various methods are known for purifying IL-2. See, for example, Current Protocols in Protein Science, Vol. 2, Eds.: John E. Coligan, Ben M. Dunn, Hidde L. Ploehg, David W. Speicher, Paul See 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 using cation exchange, gel filtration, and / or reverse-phase liquid chromatography, or from conditioned medium from either mammalian or yeast cultures producing the given mutein.

[0294] Another exemplary method for constructing a DNA sequence encoding an IL-2 mutein is by chemical synthesis. This involves the direct synthesis of a peptide by chemical means from a protein sequence encoding an IL-2 mutein exhibiting the described properties. This method can incorporate 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 selecting 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, meaning that an amino acid may 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 coded for by a single codon, TGG. Thus, for a given DNA sequence encoding a particular IL-2 mutein, it will be understood that there are many degenerate DNA sequences that encode that IL-2 mutein. For example, in addition to the preferred DNA sequence for mutein H9, the IL-2 muteins shown are: It will be understood that there are many degenerate DNA sequences that encode a particular mutein. These degenerate DNA sequences are considered to be within the scope of this disclosure. Thus, "degenerate variants thereof" in the context of the present invention refers to all DNA sequences that encode a particular mutein and thereby enable its expression.

[0295] The biological activity of the IL-2 muteins can be assayed by any suitable method known in the art, including PHA blast proliferation and NK cell proliferation.

[0296] I. Anti-PD-1 Antibodies and Combinations Anti-PD-1 antibodies for use in accordance with the inventions and methods described herein include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and the PD-1 antibodies listed in U.S. Patent Publication No. 2017 / 0081409. There are two approved anti-PD-1 antibodies, pembrolizumab (Keytruda®; MK-3475-033), and nivolumab (Opdivo®; CheckMate078), and many more in development, which can be used in the combinations described herein. Exemplary anti-PD-1 antibody sequences are shown in Figure 10, any of which can be used in the combination methods with IL-2 described herein.

[0297] In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with an anti-PD-1 antibody or inhibitor. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with nivolumab. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with pembrolizumab. In some aspects, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with cemiplimab. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with BMS-936558. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MDX-1106. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with ONO-4538. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with AMP224. In some embodiments, an IL-2 mutein containing substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with CT-011. In some embodiments, an IL-2 mutein containing substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MK-3. 475. In some embodiments, the IL-2 mutein further comprises an F42A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.

[0298] In some embodiments, an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with any of the referenced antibodies. In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. 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 90% identical to any one of SEQ ID NO:2, or SEQ ID NO:6-SEQ ID NO:10, or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, the IL-2 mutein used in combination with an anti-PD-1 antibody is a fusion mutein described herein. In some embodiments, the IL-2 mutein used in combination with an anti-PD-1 antibody is a fusion mutein described herein.

[0299] J. Anti-PD-L1 Antibodies and Combinations In some embodiments, any of the IL-2 muteins described herein can be used in combination with an anti-PD-1 antibody. There are three approved anti-PD-L1 antibodies, atezolizumab (TECENTRIQ®; MPDL3280A), avelumab (BAVENCIO®; MSB001071 8C), and durvalumab (MEDI4736), as well as other anti-PD-L1 antibodies in development. Numerous anti-PD-L1 antibodies are available, with many more in development, which can be used in combination with the anti-IL-2 muteins described herein. In some embodiments, the PD-L1 antibody is one described in U.S. Patent Publication No. 2017 / 0281764, and International Patent Publication Nos. WO2013 / 079174 (avelumab), and WO2010 / 077634 (or U.S. Patent Application No. 20160222117, or U.S. Patent No. 8,217,149; atezolizumab). In some embodiments, the PD-L1 antibody comprises the heavy chain sequence of SEQ ID NO: 34 and the light chain sequence of SEQ ID NO: 36 (from US2017 / 281764). In some embodiments, the PD-L1 antibody is atezolizumab (TECENTRIQ®; MPDL3280A; IMpower110). In some embodiments, the PD-L1 antibody is avelumab (BAVENCIO®; MSB001071 8C). In some embodiments, the PD-L1 antibody is durvalumab (MEDI4736). In some embodiments, the PD-L1 antibody includes, for example, atezolizumab (IMpower133), BMS-936559 / MDX-1105, and / or RG-7446 / MPDL3280A, and / or YW243.55.S70, and any of the exemplary anti-PD-L1 antibodies provided in Figure 11 herein. In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with any of the referenced antibodies. In some embodiments, the IL-2 mutein further comprises an F42A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO:2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, where the numbering is according to wild-type human IL-2 of SEQ ID NO:2. 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 90% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-SEQ ID NO:10 or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, the IL-2 mutein used in combination with an anti-PD-L1 antibody is a fusion mutein as described herein. In some embodiments, the IL-2 mutein used in combination with an anti-PD-L1 antibody is a fusion mutein as described herein.

[0300] K. Other immunotherapy combinations Other antibodies and / or immunotherapies for use in accordance with the methods of the invention include 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 mAbs (anti-4-1-BB antibodies) such as BMS-663513 ureumumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated by reference in their entireties); lirilumab (anti-KIR mAbs; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, e.g., U.S. Pat. Nos. 8,821,867; 8,337,850; and 9,468,678, and International Patent Application Publication No. WO 2012 / 032433, which are incorporated by reference in their entireties); anti-OX40 mAbs (see, e.g., WO 2006 / 029879 or WO 2010 / 096418, which are incorporated by reference in their entireties); anti-GITR mAbs such as TRX518 (see, e.g., U.S. Pat. No. 7,812,135, which is incorporated by reference in its entirety); anti-CD27 mAbs such as belilumab CDX-1127 Antibody antibodies include, but are not limited to, anti-ICOS mAbs (see, e.g., WO2016 / 145085 and U.S. Patent Publication Nos. US2011 / 0274685 and US2012 / 0213771, which are incorporated by reference in their entireties), anti-ICOS mAbs (e.g., MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Publication No. 2016 / 0257758, which are incorporated by reference in their entireties).In some embodiments, an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with any of the referenced antibodies. In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which follows wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, the numbering of which follows wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein is any of those disclosed herein. IL-2 muteins or variants. In some embodiments, the IL-2 mutein sequence is 90% identical to any one of SEQ ID NO:2 or SEQ ID NO:6 through SEQ ID NO:10 or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0301] Other antibodies also include monoclonal antibodies against 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, gastric cancer, and brain cancer (see generally www.clinicaltrials.gov). In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F numbered according to human wild-type IL-2 (SEQ ID NO: 2) is used in combination with any of the referenced antibodies. In some embodiments, the IL-2 mutein further contains an F42A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further contains a Y45A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further contains an E62A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. 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 90% identical to any one of SEQ ID NO:2 or SEQ ID NO:6-SEQ ID NO:10 or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16.

[0302] Antibodies also include antibodies for antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with an antibody for antibody-dependent cell-mediated cytotoxicity (ADCC). In some embodiments, the IL-2 mutein further contains an F42A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further contains a Y45A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further contains an E62A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2.

[0303] L. Treatment method In some embodiments, the subject IL-2 muteins, and / or nucleic acids expressing them, can be administered to a subject to treat disorders associated with abnormal apoptotic or differentiation processes (e.g., cell proliferative or cell differentiation disorders, such as cancer, by generating active or passive immunity). In treating such diseases, the disclosed IL-2 muteins may have advantageous properties, such as reduced 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 90% identical to SEQ ID NO:2 or any one of SEQ ID NOs:6-10 or SEQ ID NO:16. In some embodiments, the IL-2 mutein comprises any one of 5-1 SEQ ID NO:5; 5-2 SEQ ID NO:6; 6-6 SEQ ID NO:7; A2 SEQ ID NO:8; B1 SEQ ID NO:9; B11 SEQ ID NO:10; C5 SEQ ID NO:11; D10 SEQ ID NO:12; E10 SEQ ID NO:13; G8 SEQ ID NO:14; H4 SEQ ID NO:15; and H9 SEQ ID NO:16. In some embodiments, the substitutions in the IL-2 mutein are L80F, R80F, R81F, R82F, R83F, R84F, R85F, R86F, R87F, R88F, R89 ...9F, R81F, In some embodiments, the IL-2 mutein is a fusion protein. In some aspects, the IL-2 mutein is associated with 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.

[0304] Examples of cell proliferation and / or differentiation disorders include cancer (e.g., carcinoma, sarcoma, metastatic disorders, or hematopoietic neoplastic disorders, e.g., blood diseases). Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, those of prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer, including small cell lung cancer, kidney cancer, liver cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and brain cancer.

[0305] The mutant IL-2 polypeptides can be used to treat patients with, suspected of developing, or at risk of developing any type of cancer, including renal carcinoma 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 carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissue.

[0306] Additional examples of proliferative disorders include hematopoietic neoplastic disorders.

[0307] Alternatively, or in addition to methods of direct administration to a patient, in some embodiments, variant IL-2 polypeptides can be used in ex vivo methods. For example, cells (e.g., peripheral blood lymphocytes or purified populations of lymphocytes isolated from a patient and placed or maintained in culture) can be cultured in vitro in a culture medium, and the contacting step is affected by adding an IL-2 variant to the culture medium. The culturing step can include a further step of stimulating or treating the cells with other agents, for example, to stimulate proliferation or expand a cell population responsive to an antigen of interest (e.g., a cancer antigen or a viral antigen). The cells are then administered to the patient after treatment.

[0308] Anti-PD-1 antibodies for use in combination with the IL-2 muteins disclosed herein in the methods of treatment include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475.

[0309] In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with an anti-PD-1 antibody or inhibitor for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with nivolumab for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with BMS-936558 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MDX-1106 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with ONO-4538 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with ONO-4538 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with AMP224 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with CT-011 for the treatment of cancer. In some embodiments, an IL-2 mutein comprising substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with MK-3475 for the treatment of cancer. In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a K43N substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2.

[0310] In some embodiments, IL-2 muteins comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), are used in combination with anti-CTLA4 mAbs, such as ipilimumab and tremelimumab; anti-PD-L1 antagonist antibodies, such as BMS-936559 / MDX-1105, MEDI4736, and RG-7446 / MPDL3280A; anti-LAG-3 antibodies, such as IMP-321; and anti-CD40 antibodies, such as CP-870,893, lucatumumab, and dacetuzumab, for the treatment of cancer. Agonistic antibodies targeting immune stimulatory proteins, including mAbs; anti-CD137 mAbs (anti-4-1-BB antibodies) such as BMS-663513 ureumab (anti-4-1BB antibody; see, e.g., U.S. Pat. Nos. 7,288,638 and 8,962,804, which are incorporated by reference herein in their entireties); lirilumab (anti-KIR mAb; IPH2102 / BMS-986015; blocks NK cell inhibitory receptors) and PF-05082566 (utomilumab; see, e.g., U.S. Pat. Nos. 8,821,867; 8,337,850; and 9,468,678, and International Patent Application Publication No. WO2012 / 032433, which are incorporated by reference herein in their entireties); anti-OX40 mAbs (e.g., WO 2006 / 029879 or WO2010 / 096418, the entireties of which are incorporated by reference herein); anti-GITR mAbs such as TRX518 (see, e.g., U.S. Patent No. 7,812,135, the entireties of which are incorporated by reference herein); anti-CD27 mAbs such as belilumab CDX-1127 (see, e.g., WO2016 / 145085 and U.S. Patent Publication Nos. US 2011 / 0274685 and US 2012 / 0213771, the entireties of which are incorporated by reference herein); anti-ICOS mAbs such as VEGF (see, e.g., WO2016 / 145085 and U.S. Patent Publication Nos. US 2011 / 0274685 and US 2012 / 0213771, the entireties of which are incorporated by reference herein); Used in combination with antibodies and / or immunotherapies, including but not limited to mAbs (e.g., MEDI-570, JTX-2011, and anti-TIM-3 antibodies (see, e.g., WO2013 / 006490 or U.S. Patent Publication No. 2016 / 0257758, which are incorporated by reference in their entireties).

[0311] In some embodiments, an IL-2 mutein containing the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2), is used in combination with another antibody, which may include a monoclonal antibody 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), for the treatment of cancer.

[0312] In some embodiments, the IL-2 mutant includes substitutions numbered according to human wild-type IL-2 (SEQ ID NO: 2): L80F, R81D, L85V, I86V, and I92F. The antibody is used in combination with an antibody or antibodies for antibody-dependent cell-mediated cytotoxicity (ADCC) for the treatment of cancer.

[0313] M. Pharmaceutical Compositions and Methods of Administration In some embodiments, the subject IL-2 muteins and nucleic acids can be incorporated into compositions, including pharmaceutical compositions. Such compositions typically include a polypeptide or nucleic acid molecule and a pharmaceutically acceptable carrier. Such compositions can also include an anti-PD-1 antibody. In some embodiments, the composition includes an IL-2 mutein that is a fusion protein and / or associated with a CAR-T construct and / or expressed by or associated with an oncolytic virus.

[0314] The anti-PD-1 antibody and IL-2 mutein can be administered as a single composition, simultaneously as two separate compositions, and / or sequentially as two separate compositions. In some embodiments, the anti-PD-1 antibody or inhibitor and the IL-2 mutein are administered together as a single co-composition (i.e., formulated together). In some embodiments, the anti-PD-1 antibody or inhibitor and the IL-2 mutein are administered simultaneously as two separate compositions (i.e., separate formulations). In some embodiments, the anti-PD-1 antibody or inhibitor and the IL-2 mutein are administered sequentially as separate compositions (i.e., separate formulations). In some aspects, when the anti-PD-1 antibody or inhibitor and the IL-2 mutein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the IL-2 mutein. In some aspects, when the anti-PD-1 antibody or inhibitor and the IL-2 mutein are administered sequentially as separate compositions, the IL-2 mutein is administered before the anti-PD-1 antibody or inhibitor. In some embodiments, anti-PD-1 antibodies include, but are not limited to, nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475. In some embodiments, the IL-2 mutein is an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2). In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a K43N substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an F42A substitution, the numbering of which follows that of wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution, the numbering of which follows that of wild-type human IL-2 in SEQ ID NO: 2.

[0315] The other immunotherapeutic agent and the IL-2 mutein described can be administered as a single composition, simultaneously as two separate compositions, and / or sequentially as two separate compositions. In some embodiments, the other immunotherapeutic agent and the IL-2 mutein are administered together as a single co-composition (i.e., formulated together). In some embodiments, the other immunotherapeutic agent and the IL-2 mutein are administered simultaneously as two separate compositions (i.e., separate formulations). In some embodiments, the other immunotherapeutic agent and the IL-2 mutein are administered sequentially as separate compositions (i.e., separate formulations). In some aspects, when the other immunotherapeutic agent and the IL-2 mutein are administered sequentially as separate compositions, the anti-PD-1 antibody or inhibitor is administered before the IL-2 mutein. In some aspects, when the other immunotherapeutic agent and the IL-2 mutein are administered sequentially as separate compositions, the IL-2 mutein is administered before the other immunotherapeutic agent. In some aspects, the IL-2 mutein is an IL-2 mutein comprising the substitutions L80F, R81D, L85V, I86V, and I92F, numbered according to human wild-type IL-2 (SEQ ID NO: 2). In some embodiments, the IL-2 mutein further comprises the F42A substitution. and the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a K43N substitution and the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a F42A substitution and the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises a Y45A substitution and the numbering is according to wild-type human IL-2 of SEQ ID NO: 2. In some embodiments, the IL-2 mutein further comprises an E62A substitution and the numbering is according to wild-type human IL-2 of SEQ ID NO: 2.

[0316] Pharmaceutical compositions are formulated to be compatible with their intended route of administration. While anti-PD-1 antibodies and / or mutant IL-2 polypeptides of the present invention may be administered orally, they are more likely to be administered parenterally, including intravenously. Examples of parenteral routes include intravenous, intradermal, subcutaneous, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral administration contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and a tonicity adjuster such as sodium chloride or dextrose. The pH can be adjusted (e.g., to about 7.2-7.8, e.g., 7.5) with an acid or base, such as monobasic and / or dibasic sodium phosphate, hydrochloric acid, or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0317] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble), or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. 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. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants, such as sodium dodecyl sulfate. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. 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 injectable compositions can be achieved by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0318] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the ingredients listed above as necessary, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle, and sterile vehicle contains basic dispersion medium and other necessary ingredients listed above.For the preparation of sterile injectable solution, the preferred method of preparing sterile powder is vacuum drying and freeze-drying, which can obtain the powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0319] Oral compositions, if used, generally include an inert diluent or an edible carrier. For administration purposes, the active compound can be mixed with excipients and used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose, disintegrating agents such as alginic acid, Primogel®, or cornstarch; lubricants such as magnesium stearate or Sterotes®; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; and flavorings such as peppermint, methyl salicylate, or orange flavor.

[0320] For administration by inhalation, the anti-PD-1 antibodies and / or IL-2 muteins, or nucleic acids encoding them, are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U.S. Patent No. 6,468,798.

[0321] Systemic administration of anti-PD-1 antibodies and / or IL-2 muteins or nucleic acids may be via transmucosal or transdermal routes. 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, surfactants, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, medicated ointments, gels, or creams as are generally known in the art.

[0322] In some embodiments, the compounds (anti-PD-1 antibodies and / or 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.

[0323] In some embodiments, the compound (subject IL-2 mutein or nucleic acid) can also be administered by transfection or infection using methods known in the art, including, but not limited to, those described in McCaffrey et al. (Nature 418:6893, 2002), Xia et al. (Nature Biotechnol. 20:1006-1010, 2002), or Putnam (Am. J. Health Syst. Pharm. 53:151-160, 1996, erratum at Am. J. Health Syst. Pharm. 53:325, 1996).

[0324] In one embodiment, the anti-PD-1 antibody and / or IL-2 mutein or nucleic acid is prepared with a carrier that protects the anti-PD-1 antibody and / or 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 using 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.

[0325] Dosage, toxicity, and therapeutic efficacy of such anti-PD-1 antibodies, IL-2 muteins, or nucleic acid compounds can be determined by standard pharmaceutical techniques in cell cultures or experimental animals, e.g., by LD 50 (50% lethal dose of the population) or ED 50 To determine the dose that is therapeutically effective in 50% of the population, the LD can be determined. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is defined as the LD 50 / ED 50 The therapeutic index can be expressed as a ratio of . Compounds that exhibit a high therapeutic index are preferred. Compounds that exhibit toxic side effects may be used, but care must be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells, thereby reducing side effects.

[0326] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for human use. The dose of such compounds can be adjusted to EDTA with little or no toxicity. 50 Preferably, the circulating concentration of the compound is within a range including the IC50, IC60, IC70, IC80, IC90, IC10, IC11, IC12, IC13, IC14, IC15, IC26, IC27, IC28, IC29, IC30, IC40, IC51, IC61, IC72, IC83, IC94, IC105, IC116, IC126, IC136, IC146, IC157, IC16, IC27, IC28, IC29, IC30, IC166, IC29, IC29, IC30, IC40, IC51, IC61, IC126, IC29, IC29, IC29, IC30, IC40, IC51, IC61, IC146, IC29, IC29, IC29, IC30, IC29, IC29, IC30, IC40, IC51, IC61, IC29, IC29, IC29, IC30 ...30, IC40, IC51, IC61, IC29, IC29, IC29, IC30, IC29, IC30, IC40, IC51, IC61, IC29, IC29, IC2 50 A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the concentration of the test compound that achieves a half-maximal inhibition of symptoms (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0327] As defined herein, the therapeutically effective amount (i.e., effective dose) of a subject IL-2 mutein and / or anti-PD-1 antibody or inhibitor will depend on the polypeptide or antibody selected. In some embodiments, a single dose of an IL-2 mutein may range from about 0.001 mg / kg to 0.1 mg / kg per kg of patient body weight to be administered. In some embodiments, a single dose of an anti-PD-1 antibody or inhibitor may range from about 1 mg / kg to 20 mg / kg, or from about 5 mg / kg to about 15 mg / kg, or about 10 mg / kg per kg of patient body weight to be administered. In some embodiments, doses of an anti-PD-1 antibody or inhibitor and / or IL-2 mutein of about 0.005 mg / kg, 0.01 mg / kg, 0.025 mg / kg, 0.05 mg / kg, 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 5.0 mg / kg, or 10.0 mg / kg may be administered. In some embodiments, 600,000 IU / kg is administered (IU can be determined by lymphocyte proliferation bioassay and is expressed in International Units (IU) established by the World Health Organization's First International Standard for Interleukin-2 (human)). Dosages can be equivalent to, but are expected to be less than, those prescribed for PROLEUKIN®. The composition can be administered one or more times daily to one or more times weekly, including once every other day. Those skilled in the art will appreciate 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 a subject IL-2 mutein can include a single treatment or can include a series of treatments. In one embodiment, the composition is administered every 8 hours for 5 days, followed by a 2-14 day, e.g., 9-day, rest period, followed by administration every 8 hours for another 5 days. In some embodiments, administration is three times every 4 days.

[0328] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0329] The following examples are provided to illustrate certain embodiments of the invention provided herein and should not be construed as limiting. Illustrative Embodiments 1. A method of treating cancer, comprising: (i) an anti-PD-1 antibody or inhibitor, or an anti-PD-L1 antibody or inhibitor; (ii) an IL-2 mutein comprising the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, the numbering of which is according to wild-type human IL-2 of SEQ ID NO: 2. 2. The method of claim 1, wherein the anti-PD-1 antibody or inhibitor is selected from the group consisting of nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and the PD-1 antibodies listed in U.S. Patent Publication No. 2017 / 0081409. 3. The method of claim 1, wherein the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. 4. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 5. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises a K43N substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 6. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 7. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises a Y45A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 8. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an E62A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 9. The method of any one of claims 1 to 8, wherein the IL-2 mutein is a fusion protein. 10. The method of claim 9, wherein the fusion protein comprises the IL-2 linked to an Fc antibody fragment. 11. The method of claim 10, wherein the Fc antibody fragment is a human Fc antibody fragment. 12. The method of claim 10, wherein the Fc antibody fragment comprises an N297A substitution. 13. The method of claim 9, wherein the fusion protein comprises the IL-2 linked to albumin. 14. The method of any one of claims 1 to 13, wherein the cancer is selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, kidney cancer, liver cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and brain cancer. 15. The method of claim 14, wherein the cancer is colon cancer. 16. The method of any one of claims 1 to 15, wherein the IL-2 mutein exhibits increased binding ability to IL-2Rβ compared to wild-type human IL-2. 17. The method of any one of claims 1 to 15, wherein the IL-2 mutein exhibits a higher binding affinity to IL-2Rβ compared to wild-type human IL-2. 18. The method of any one of claims 4 to 8, wherein the IL-2 mutein exhibits reduced binding affinity for CD25 compared to wild-type human IL-2. 19. A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, an IL-2 mutein according to any one of claims 1 to 13 or 16 to 18, and a pharmaceutically acceptable carrier. 20. An immune cell targeting or expression construct comprising an interleukin-2 receptor beta (IL-2Rβ) binding protein, wherein the equilibrium dissociation constant of said binding protein with IL-2Rβ is less than that of wild-type human IL-2 (hIL-2), linked to an immune cell targeting or expression construct comprising at least one other targeting moiety. 21. The construct of claim 20, wherein the immune cell targeting or expression construct exhibits a cytotoxic effect on T cells, e.g., CD8+ T cells or CD4+ T cells. 22. The construct of any one of claims 20 to 21, wherein the construct is a chimeric antigen receptor (CAR) and the IL-2 mutein is fused to a transmembrane domain and linked to an intracellular signaling region. 23. The construct of claim 22, wherein the intracellular signaling region comprises a CD3 signaling domain. 24. The construct of claim 23, wherein the intracellular signaling region comprises one or more of a CD28 signaling domain, a CD137 signaling domain, an OX-40 signaling domain, an ICOS signaling domain, and a DAP10 signaling domain. 25. The construct of claim 21, wherein the construct is a T cell antigen conjugate (TAC) and the IL-2 mutein or other targeting moiety is fused to a ligand that binds to a protein associated with the TCR complex; or to a T cell receptor signaling domain polypeptide. 26. The construct of claim 25, wherein the protein associated with the TCR complex is CD3. 27. The construct of claim 26, wherein the T cell receptor signaling domain polypeptide comprises a CD4 cytosolic domain and a CD4 transmembrane domain. 28. The construct of claim 20, wherein the construct is an antibody-binding T-cell receptor (ACTR) comprising a chimeric antigen receptor component that binds with high affinity to an IL-2 mutein or other targeting moiety. 29. The construct of claim 20, wherein the CAR component comprises CD16 and the IL-2 mutein is fused to an Fc sequence. 30. The construct of claim 20, wherein the construct is a bispecific T cell exchanger (BiTE) comprising an IL-2 mutein or other targeting moiety fused to a variable region of an antibody that binds to a component of the T cell receptor. 31. The construct of claim 30, wherein the BiTE component of the T cell receptor is CD3. 32. The construct of claim 31, wherein the IL-2Rβ binding protein comprises the following amino acid substitutions numbered according to wild-type hIL-2: L80F, R81D, L85V, I86V, and I92F. 33. A nucleic acid encoding the construct according to any one of claims 20 to 32. 34. A vector comprising the nucleic acid construct of claim 33. 35. A T cell comprising the construct of claim 33 or the vector of claim 34. 36. A NK cell comprising the construct of claim 33 or the vector of claim 34. 37. T cells are CD4 + 36. The T cell of claim 35, which is a T cell. 38. T cells are CD8 + 36. The T cell of claim 35, which is a T cell. 39. An isolated population of immune cells according to claim 35 or claim 36. 40. A pharmaceutical formulation comprising the immune cell population of claim 39. 41. A method of targeting cancer cells, including cells that express the IL-2 receptor, comprising contacting the cells with the formulation of claim 40. 42. The method of claim 41, wherein said contacting is in vitro. 43. The method of claim 41, wherein said contacting is in vivo. 44. A method of treating cancer, comprising contacting an individual having cancer with an effective amount of the formulation of claim 40. 45. The method of claim 44, wherein the cancer is leukemia, lymphoma, glioblastoma, medulloblastoma, breast cancer, head and neck cancer, renal cancer, ovarian cancer, Kaposi's sarcoma, acute myeloid leukemia, B-lineage malignancies, colorectal, pancreatic, renal, or mesothelioma. 46. ​​A method for targeting an IL-2 mutein protein to cancer cells, comprising contacting the cancer cells with an IL-2 mutein oncolytic virus combination, wherein the combination comprises an IL-2 mutein conjugated to or expressed by an oncolytic virus, and the oncolytic virus is capable of targeting the cancer cells. 47. The method of claim 46, wherein said contacting occurs in vitro. 48. The method of claim 46, wherein said contacting occurs in vivo. 49. The method of any one of claims 46 to 48, wherein the oncolytic virus is selected from the group consisting of adenovirus, autonomously replicating alphavirus, vaccinia virus, Seneca Valley virus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus (VSV), herpesvirus (including HSV-1 and HSV-2), measles virus, poliovirus, reovirus, coxsackie virus, lentivirus, morbillivirus, influenza virus, Sinbis virus, myxoma virus, and retrovirus. 50. The method of claim 47, wherein the vaccinia virus genome comprises a thymidine kinase gene (TK) inactivated by an open reading frame excising a deletion of at least one nucleotide providing a replacement in the gene and / or a partially deleted thymidine kinase gene, the vaccinia growth factor gene is deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding an IL-2 mutein described herein. 51. The method of claim 48, wherein said in vivo contacting results in an increase in the concentration of the IL-2 mutein protein in the tumor microenvironment compared to the concentration of the IL-2 mutein protein that is not conjugated to an oncolytic virus. 52. The method of any one of claims 46 or 48-51, wherein the modified oncolytic virus targets the IL-2 mutein to immunosuppressive cells of the tumor microenvironment (TME), such as tumor-associated macrophages and MDSCs (myeloid-derived suppressor cells), to improve therapeutic benefit. 53. The method of any one of claims 46 or 48-52, wherein the modified oncolytic virus targets the IL-2 mutein to one or more immunosuppressive cells that express one or more tumor antigens. 54. The method of any one of claims 46 or 48-53, wherein the modified oncolytic virus targets an IL-2 mutein to the TME. 55. The method of any one of claims 46 or 48-54, wherein the IL-2 mutein protein enhances effector T cells and / or NK cells. 56. The method of any one of claims 46 or 48-55, wherein the IL-2 mutein suppresses Treg activity. 57. The method of any one of claims 46 or 48-56, wherein the IL-2 comprises the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, the numbering of which is according to wild-type human IL-2 in SEQ ID NO: 2. 58. A modified vaccinia virus vector, characterized in that the vector comprises a vaccinia virus genome, the thymidine kinase gene of which has been inactivated by a replacement in the thymidine kinase gene (TK) gene and / or an open reading frame excising a deletion of at least one nucleotide providing a partially deleted thymidine kinase gene, the vaccinia growth factor gene has been deleted, and the modified vaccinia virus vector comprises at least one nucleic acid sequence encoding an IL-2 mutein as described herein. 59. A modified nucleic acid comprising: (i) a modified nucleic acid, optionally having nucleotides encoding amino acids 122-129 of the encoded E1A polypeptide deleted; and (ii) an expression cassette comprising a polynucleotide encoding an IL-2 mutein described herein. Oncolytic adenovirus. 60. The modified virus of claim 58 or claim 59, wherein the IL-2 mutein directs the modified oncolytic virus to immunosuppressive cells of the tumor microenvironment (TME), such as tumor-associated macrophages and MDSCs (myeloid-derived suppressor cells), for improved therapeutic benefit. 61. The modified virus of claim 58 or claim 59, wherein the IL-2 mutein protein directs the modified oncolytic virus to one or more tumor antigens. 62. The modified virus of claim 58 or claim 59, wherein the IL-2 mutein protein directs the modified oncolytic virus to the TME. 63. The modified virus of claim 58 or claim 59, wherein the IL-2 mutein protein enhances effector T cells and NK cells. 64. The modified virus of claim 58 or claim 59, wherein the IL-2 mutein suppresses Treg activity. 65. A method of treating cancer comprising administering to a subject in need thereof an oncolytic virus capable of expressing an IL-2 mutein. 66. The method of claim 65, wherein the IL-2 mutein comprises the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:2. 67. The method of any one of claims 65-66, wherein the oncolytic virus is selected from the group consisting of adenovirus, autonomously replicating alphavirus, vaccinia virus, Seneca Valley virus, Newcastle disease virus, Maraba virus, vesicular stomatitis virus (VSV), herpesvirus (including HSV-1 and HSV-2), measles virus, poliovirus, reovirus, coxsackievirus, lentivirus, morbillivirus, influenza virus, Simbis virus, myxoma virus, and retrovirus. [Example]

[0330] Example 1: H9 synergizes with anti-PD-1 immunotherapy in a murine MC38 colon cancer model. This example provides data showing that combination therapy produces robust responses in a dose-dependent manner.

[0331] Table 11 below shows the substitution matrix for the H9 IL-2 mutein used in this example. [Table 11]

[0332] Anti-PD-1 antibodies were administered intravenously at 10 mg / kg, three times every four days (10 mg / kg IV q4dx3). H9 (an IL-2 mutein with amino acid substitutions L80F, R81D, L85V, I86V, and I92F, numbering follows that of wild-type human IL-2 in SEQ ID NO: 2) was administered at the indicated doses of 5 μg q.d. or 25 μg q.d. (dosage μg / mouse) according to the same dosing regimen. MC38 colon cancer model mice were then monitored for up to 40 days after tumor implantation. The combination of anti-PD-1 antibodies and H9 increased the number of cured mice at both low and high doses, with a 25 μg q.d. dose of H9 demonstrating improved efficacy. increased substantially.

[0333] As shown in the data in Figure 1, H9 and anti-PD-1 provide only limited efficacy. However, the combination therapy is sufficient to cure most mice at well-tolerated H9 doses. The increased efficacy of the combination did not result in new or increased toxicity.

[0334] The above examples are provided to provide those of skill in the art with a complete disclosure and description of how to make and use embodiments of the compositions, systems, and methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention. Modifications of the above-described modes for carrying out the invention that are obvious to those of skill in the art are intended to be within the scope of the appended claims. All patents and publications mentioned herein are indicative of the level of skill of those skilled in the art to which this invention pertains. All references cited in this disclosure are incorporated by reference to the same extent as if each reference was individually incorporated by reference in its entirety.

[0335] All heading and section designations are used for clarity and reference purposes only and should not be considered limiting in any way. For example, those skilled in the art will recognize the utility of combining various aspects from different headings and sections as appropriate in accordance with the spirit and scope of the invention described herein.

[0336] All references cited in this specification are incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0337] It will be apparent to those skilled in the art that many modifications and variations of this application can be made without departing from the spirit and scope thereof. The specific embodiments and examples described herein are offered by way of example only and should be 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. A method of treating cancer, comprising: (i) an anti-PD-1 antibody or inhibitor, or an anti-PD-L1 antibody or inhibitor; (ii) an IL-2 mutein comprising the following amino acid substitutions: L80F, R81D, L85V, I86V, and I92F, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

2. 2. The method of claim 1, wherein the anti-PD-1 antibody or inhibitor is selected from the group consisting of nivolumab, BMS-936558, MDX-1106, ONO-4538, AMP224, CT-011, and MK-3475 (pembrolizumab), cemiplimab (REGN2810), SHR-1210 (CTR20160175 and CTR20170090), SHR-1210 (CTR20170299 and CTR20170322), JS-001 (CTR20160274), IBI308 (CTR20160735), BGB-A317 (CTR20160872), and the PD-1 antibodies listed in U.S. Patent Publication No. 2017 / 0081409.

3. 2. The method of claim 1, wherein the anti-PD-L1 antibody or inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.

4. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

5. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises a K43N substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

6. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an F42A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

7. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises a Y45A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

8. The method of any one of claims 1 to 3, wherein the IL-2 mutein further comprises an E62A substitution, the numbering of which is according to wild-type human IL-2 of SEQ ID NO:

2.

9. The method of any one of claims 1 to 8, wherein the IL-2 mutein is a fusion protein.

10. 10. The method of claim 9, wherein the fusion protein comprises the IL-2 linked to an Fc antibody fragment.

11. The method of claim 10, wherein the Fc antibody fragment is a human Fc antibody fragment.

12. 11. The method of claim 10, wherein the Fc antibody fragment comprises an N297A substitution.

13. 10. The method of claim 9, wherein the fusion protein comprises the IL-2 linked to albumin.

14. The cancer is selected from the group consisting of prostate cancer, ovarian cancer, breast cancer, endometrial cancer, multiple myeloma, melanoma, lymphoma, lung cancer including small cell lung cancer, kidney cancer, liver cancer, colon cancer, colorectal cancer, pancreatic cancer, gastric cancer, and brain cancer. The method of any one of claims 1 to 13, wherein the cancer is selected from the group consisting of:

15. 15. The method of claim 14, wherein the cancer is colon cancer.

16. The method of any one of claims 1 to 15, wherein the IL-2 mutein exhibits increased binding ability to IL-2Rβ compared to wild-type human IL-2.

17. The method of any one of claims 1 to 15, wherein the IL-2 mutein exhibits a higher binding affinity to IL-2Rβ compared to wild-type human IL-2.

18. The method of any one of claims 4 to 8, wherein the IL-2 mutein exhibits a reduced binding affinity to CD25 compared to wild-type human IL-2.

19. A pharmaceutical composition comprising an anti-PD-1 antibody or inhibitor, an IL-2 mutein according to any one of claims 1 to 13 or 16 to 18, and a pharmaceutically acceptable carrier.

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