Fusions of mutant interleukin-2 polypeptides with antigen binding molecules for modulating immune cell function

Abstract

To provide fusions of mutant interleukin-2 polypeptides with antigen binding molecules for modulating immune cell function.SOLUTION: Provided herein are fusion proteins that bind human CD8α, human CDSβ, or human PD1 and comprise a mutant IL-2 polypeptide, as well as polynucleotides, host cells, compositions, and methods of use thereof. The present disclosure describes, inter alia, mutant IL-2 polypeptides, where the mutant IL-2 polypeptides have one, two or more, or three or more amino acid substitutions (i.e. mutations) relative to the wild-type mature IL-2 amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 857,726, filed June 5, 2019, which is incorporated by reference in its entirety.

[0002] Submitting a sequence listing as an ASCII text file The following submission in an ASCII text file is incorporated by reference in its entirety: Sequence Listing in Computer Readable Format (CRF) (Filename: 182842000140SEQLIST.TXT, Recorded: June 5, 2020, Size: 105KB).

[0003] The present disclosure discloses mutant interleukin-2 polypeptides and fusion polypeptides comprising mutant interleukin-2 polypeptides and antigen-binding molecules. The present disclosure provides a method for modulating immune cell function by contacting immune cells with the fusion polypeptides of the present disclosure. In addition, the present disclosure also provides polynucleotides encoding the fusion proteins of the present disclosure, as well as vectors and host cells comprising such polynucleotides. The present disclosure further provides methods for producing the fusion proteins, pharmaceutical compositions comprising the fusion proteins, and their uses. [Background technology]

[0004] Interleukin-2 (IL-2) is a cytokine that regulates many lymphocyte subsets, including alpha beta CD4+ and CD8 T+ cells, as well as various innate and innate-like lymphocytes, such as NK cells, NK T cells, gamma delta T cells (Tγδ) cells, and innate lymphoid cells (ILC1, ILC2, and ILC3 cells). Binding of IL-2 to its receptor induces phosphorylation of the receptor-associated Janus kinases, JAK3 and JAK1, which promote phosphorylation of the STAT5 transcription factor (pSTAT5), which regulates the transcription of many genes in lymphocytes. Binding of IL-2 to its receptor also activates other signaling pathways, in addition to STAT5, such as ERK, PI3K, and Akt kinases. IL-2 signaling in lymphocytes promotes cell survival, proliferation, and enhanced effector functions, including proinflammatory cytokine secretion and cytotoxic function, and in some cases, activation-induced cell death (reviewed in Ross & Cantrell, Annu Rev Immunol. 2018 Apr 26;36:411-433).

[0005] IL-2 can signal by binding with moderate affinity to a receptor complex consisting of the IL-2Rβ and IL-2Rγ subunits (IL-2Rβγ, intermediate affinity receptor), both of which are required and sufficient to trigger downstream signaling in immune cells. In addition, IL-2 binds with high affinity to a receptor complex consisting of the IL-2Rα, IL-2Rβ, and IL-2Rγ subunits (IL-2Rαβγ, high affinity receptor) (Stauber et al, Proc Natl Acad Sci US A. 2006 Feb 21;103(8):2788-93). IL-2Rα expression is associated with increased expression of CD4+ T cells. IL-2 is restricted to reg cells, activated T lymphocytes, and ILC2 and ILC3 cells, making these subsets the most sensitive to IL-2 signaling. The IL-2Rβ and IL-2Rγ subunits are shared with another related cytokine, IL-15, and the IL-2Rγ subunit is shared among other common gamma chain cytokines (IL-4, IL-7, IL-9, and IL-21). NK cells, NK Many innate and innate-like lymphocytes, including T cells, Tγδ cells, and ILC1, ILC2, and ILC3 cells, express high levels of IL-2Rβ (ImmGen consortium; Heng TS et al, Immunological Genome Project Consortium. Nat Immunol. 2008 Oct;9(10):1091-4), which also identifies such lymphocytes as IL-2 receptors. This renders the antibody sensitive to both IL-1 and IL-15 cytokines.

[0006] Consistent with its potent activity on lymphocytes, systemic administration of high doses of IL-2 resulted in the activation and efficacy of antitumor immune responses in many preclinical cancer models. Systemically administered high dose IL-2 has also been tested in patients, with high doses of IL-2 approved for the treatment of metastatic melanoma and renal cell carcinoma (RCC). The dosing regimen consisted of intravenous injections of 600,000 IU / kg every 8 hours, established based on the in vivo half-life of IL-2 to maintain serum levels at the concentration required to stimulate the high affinity IL-2 receptor. The overall response rate in RCC was 20% with a complete response rate of 9%, while the overall response rate in melanoma was 16% with a complete response rate of 6% (reviewed in Rosenberg, J Immunol.2014 Jun 15;192(12):5451-8). High-Dose IL-2 in Cancer The efficacy of IL-2 is thought to be due to its ability to robustly expand T and NK cells while maintaining their function. However, IL-2 also expands Treg cells and promotes their proper suppressive function (Chinen et al, Nat Immunol. 2016 Nov; 17(11): 1322-1333). Indeed, due to the sensitivity of Tregs to IL-2, low-dose IL-2 treatment regimens are being tested in patients with autoimmunity to suppress pathogenic immune responses (Collison, Nat Rev Rheumatol. 2019 Jan; 15(1): 2).

[0007] In addition to its undesirable effect on immunosuppressive Treg cells, the benefits of IL-2 in patients were accompanied by significant toxicity, including fever, chills, fatigue, joint pain, hypotension, abnormal liver function, renal failure, as well as capillary leak syndrome and fluid retention.IL-2 treatment requires strict patient eligibility criteria and administration by experienced physicians, because the toxicity induced by IL-2 limits the number of doses that patients may take (Schwartz et al, Oncology (Williston Park).2002 Nov;16(11 Suppl 13):11-20). IL-2 toxicity involves a complex set of interactions between immune cells and vascular endothelium: IL-2-activated cells bind tightly to endothelial cells, resulting in their lysis, and IL-2 induces pulmonary edema by its interaction with functional IL-2 receptors on endothelial cells (reviewed in Milling et al, Adv Drug Deliv Rev. 2017 May 15; 114: 79-101). Blockade of IL-2 interaction with IL-2Rα abolished pulmonary edema in animal models (Krieg et al, Proc Natl Acad Sci US A. 2010 Jun 29;107(26):11906-11 In addition, the study showed that blockade of IL-2Rα also led to robust activation of IL-2Rβγ+ effector immune cells, CD8+ T cells and NK cells, and to a lesser extent, Tregs, resulting in significant improvements in both safety and antitumor efficacy compared to recombinant IL-2.

[0008] Recently, NK cells have been shown to cause IL-2 toxicity in mice by NK cell hyperactivation and secretion of multiple inflammatory cytokines when IL-2 is administered together with IFN-α (Rothschilds et al, Oncoimmunology.2019 Feb 19;8(5):e1558678). In addition, NK cells have also been shown to cause toxicity of the cytokine IL-15, which also signals through IL-2Rβγ (Guo et al, J Immunol.2015 Sep 1;195(5):2353-64). This NK cell hyperactivation in response to IL-2Rβγ signaling is likely due to NK cells' high expression of IL-2Rβ and their ability to rapidly secrete inflammatory cytokines in response to activation. In addition, other innate lymphocytes that also express high levels of IL-2Rβ may also contribute to the systemic toxicity observed with systemic administration of IL-2, although their role in inducing IL-2 toxicity has not been studied.

[0009] On the other hand, CD8+ T cells have been shown to mediate the efficacy of immunotherapeutic agents, including IL-2, in many preclinical cancer models (Caudana et al, Cancer Immunol Res. 2019 Mar;7(3):443-457), and they correlate with response to immunotherapy in patients. (Sade-Feldman et al, Cell.2018 Nov 1;175(4):998-1013). CD8+ T cells express CD8, a type I transmembrane glycoprotein found on the cell surface as CD8 alpha (CD8α, CD8a) homodimers and CD8 alpha-CD8 beta (CD8β, CD8b) heterodimers. CD8 dimers interact with major histocompatibility (MHC) class I molecules on target cells, and this interaction mediates the TCR's identification of CD8 + It is kept in tight engagement with MHC during T cell activation. The cytoplasmic tail of CD8α contains a binding site for T cell kinase (Lck), which initiates signaling downstream of the TCR during T cell activation, while the role of CD8β is thought to be to increase the avidity of CD8 binding to MHC class I and to influence the specificity of the CD8 / MHC / TCR interaction (Bosselut et al, Immunity. 2000 Apr;12(4):409-18).

[0010] Intratumoral T cells have recently been shown to express PD1 in several human cancers (Gros et al, J Clin Invest. 2014 May;124(5):2246-59;Egelston etl al, Nat Commun. 2018 Oct 16;9(1):4297;Thommen et al, Nat Med. 2018 Jul;24(7):994-1004). PD1 is an I-protein complex that contains an extracellular domain, a transmembrane region, and a cytoplasmic tail. PD1 is a type I transmembrane protein. The cytoplasmic tail contains phosphorylation sites that are part of immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that can recruit intracellular phosphatases such as SHP-1 and SHP-2. PD1 negatively regulates TCR signaling by binding to its ligands PD-L1 and PD-L2. This interaction of PD1 with its ligands is blocked by several approved anti-PD1 and anti-PD-L1 antibodies as cancer treatments (Ribas & Wolchok, Science.2018 Mar 23;359(6382):1350-1355).

[0011] High expression of PD1 on intratumoral T cells correlates with specificity for tumor antigens, and the frequency of these PD1+ T cells in tumors correlates with response to anti-PD1 antibodies (Thommen et al, Nat Med.2018 Jul;24(7):994-1004). PD1 is expressed in peripheral blood PD1 is also expressed on CD8+ and CD4+ memory and effector T cells, and may also be expressed on T cells present in healthy tissues, although at lower levels than on tumor antigen-specific intratumoral T cells. In addition, other cell types, such as Treg, Tγδ, NK T, and ILC2 cells, may also express PD1. [Prior art documents] [Non-patent literature]

[0012] [Non-Patent Document 1] Ribas and Wolchok, Science (2018) 359(6382):1350-1355 [Non-Patent Document 2] Thommen et al., Nat Med. (2018) 24(7):994~1004 Summary of the Invention [Problem to be solved by the invention]

[0013] The goal is to reduce the toxicity and improve the efficacy of IL-2 by enhancing its activity against CD8+ or PD1+ T cells, which has been associated with efficacy in preclinical cancer models and in cancer patients, and by reducing its activity against other cells, including Tregs and innate lymphoid cells, which have been associated with IL-2 toxicity and unwanted effects. [Means for solving the problem]

[0014] The present disclosure describes, inter alia, mutant IL-2 polypeptides having one, two or more, or three or more amino acid substitutions (i.e., mutations) relative to the wild-type mature IL-2 amino acid sequence, e.g., as depicted in Figure 1A (SEQ ID NO:1) and Figure 2. In some embodiments, the mutant IL-2 polypeptides exhibit reduced binding affinity to an IL-2Rα polypeptide having the amino acid sequence depicted in Figure 1B (SEQ ID NO:2) compared to the binding affinity of the wild-type IL-2 polypeptide. In some embodiments, the mutant IL-2 polypeptides exhibit reduced binding affinity to an IL-2Rα polypeptide having the amino acid sequence depicted in Figure 1B (SEQ ID NO:2) compared to the binding affinity of the wild-type IL-2 polypeptide, and exhibit reduced binding affinity to an IL-2Rβ polypeptide having the amino acid sequence depicted in Figure 1C (SEQ ID NO:3) compared to the binding affinity of the wild-type IL-2 polypeptide. In some embodiments, the mutant IL-2 polypeptides exhibit reduced binding affinity for an IL-2Rα polypeptide having the amino acid sequence depicted in FIG. 1B (SEQ ID NO:2) compared to the binding affinity of the wild-type IL-2 polypeptide, and exhibit reduced binding affinity for an IL-2Rγ polypeptide having the amino acid sequence depicted in FIG. 1D (SEQ ID NO:4) compared to the binding affinity of the wild-type IL-2 polypeptide. In some embodiments, the mutant IL-2 polypeptides exhibit reduced binding affinity for an IL-2Rα polypeptide having the amino acid sequence depicted in FIG. 1B (SEQ ID NO:2) compared to the binding affinity of the wild-type IL-2 polypeptide, exhibit reduced binding affinity for an IL-2Rβ polypeptide having the amino acid sequence depicted in FIG. 1C (SEQ ID NO:3) compared to the binding affinity of the wild-type IL-2 polypeptide, and exhibit reduced binding affinity for an IL-2Rγ polypeptide having the amino acid sequence depicted in FIG. 1D (SEQ ID NO:4) compared to the binding affinity of the wild-type IL-2 polypeptide. In some embodiments, the mutant IL-2 polypeptides exhibit improved biophysical properties compared to the wild-type IL-2 polypeptide.

[0015] The mutant IL-2 polypeptides disclosed herein have a reduced ability to bind to and / or stimulate immune cells associated with undesirable effects of IL-2 on efficacy, such as Tregs, or immune cells associated with toxicity of IL-2, such as innate lymphoid cells, including NK cells, compared to wild-type IL-2, due to their reduced binding affinity to the IL-2R complex. However, the mutant IL-2 polypeptides of the present disclosure also have a reduced ability to bind to and / or activate desirable IL-2R-expressing immune cells, such as CD8+ T cells, which are associated with efficacy in preclinical cancer models and response to immunotherapy in patients, compared to wild-type IL-2. To make the mutant IL-2 polypeptides of the present disclosure into therapeutic agents that may be both safer and more effective for the treatment of cancer and other immune-related diseases, such as certain infectious diseases, the inventors have designed fusion proteins comprising the mutant IL-2 polypeptides of the present disclosure and antigen-binding molecules, such as antibodies against antigens present on CD8+ T cells, such as CD8 and PD1. Such fusion proteins comprising a mutant IL-2 polypeptide and an antibody that binds to a specific antigen are also called "targeted" fusion proteins, since they bind to the antigen recognized by the antigen-binding molecule of the fusion. Such fusion proteins are distinguished from "non-targeted" fusion proteins comprising a mutant IL-2 polypeptide and a control antibody that does not bind to any specific antigen (i.e., Fc fusion with an IL-2 polypeptide or a control antibody fusion; Zhu et al, Cancer Cell. 2015 Apr 13; 27(4): 489-501).

[0016] Without wishing to be bound by theory, FIG. 3 depicts a general mechanism of how an antigen binding molecule that binds to an antigen on a CD8+ T cell works to increase binding and / or stimulation of a mutant IL-2 polypeptide to a CD8+ T cell in the context of a targeted fusion protein of the present disclosure that contains said mutant IL-2 polypeptide. Certain antigen binding molecules, when fused to a mutant IL-2 polypeptide, are capable of significantly increasing binding and / or significantly increasing activity of the mutant IL-2 polypeptide only to cells that express an antigen to the antigen binding molecule of the fusion, thereby preferentially activating antigen-expressing cells over non-antigen-expressing cells (FIG. 3). Unlike targeted fusion proteins, non-targeted fusion proteins containing the same mutant IL-2 polypeptide do not preferentially bind and / or activate antigen-expressing cells (FIG. 3).

[0017] Without wishing to be bound by theory, it is believed that the difference between activation of antigen-expressing cells by targeted fusion proteins and activation of non-antigen-expressing cells, and between activation of antigen-expressing cells by targeted fusion proteins and activation of antigen-expressing cells by non-targeted fusion proteins, is important to the efficacy of the targeted fusion protein as a therapeutic agent, and these differences can be measured experimentally. Fusion proteins that are more selective for cells associated with efficacy, such as CD8+ T cells, over other cells associated with toxicity or undesirable effects on efficacy may have a higher therapeutic index when the fusion protein is used as a therapeutic agent.

[0018] Certain aspects of the present disclosure relate to fusion proteins comprising two moieties. In some embodiments, the first moiety comprises an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer (wherein VH is the variable heavy chain and CH2-CH3 is the Fc domain), an antibody light chain VL-CL (wherein VL is the variable light chain and CL is the constant light chain), and a mutant IL-2 polypeptide, the N-terminus of the mutant IL-2 polypeptide being fused to the C-terminus of the Fc domain via a linker; the second moiety comprises an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; and both the first moiety and the second moiety bind to an epitope on one antigen selected from the following group: human CD8α, human CD8β, and human PD1. In some embodiments, the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer (wherein CH2-CH3 is an Fc domain) and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; and the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1. In some embodiments, the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer (wherein CH2-CH3 is an Fc domain) and a mutant IL-2 polypeptide, where the C-terminus of the mutant IL-2 polypeptide is fused to the N-terminus of the Fc domain via a linker; the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1. In some embodiments, the first portion comprises an antigen binding domain that binds to human CD8α or human CD8β; the second portion comprises a mutant IL-2 polypeptide; the second portion is linked to the first portion via a linker (e.g., the second portion is fused to the first portion).

[0019] In some embodiments, the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1, and a 50% or greater reduction in binding affinity to an IL-2Rβ polypeptide having the amino acid sequence of SEQ ID NO:3 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1, and a 50% or greater reduction in binding affinity to an IL-2Rγ polypeptide having the amino acid sequence of SEQ ID NO:4 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide exhibits a 50% or more reduced binding affinity to an IL-2R alpha polypeptide having the amino acid sequence of SEQ ID NO: 2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 1, a 50% or more reduced binding affinity to an IL-2R beta polypeptide having the amino acid sequence of SEQ ID NO: 3 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 1, and a 50% or more reduced binding affinity to an IL-2R gamma polypeptide having the amino acid sequence of SEQ ID NO: 4 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO: 1.

[0020] In some embodiments, the binding affinity of a mutant IL-2 polypeptide to IL-2Rα is measured by comparing the activation of Treg cells by a fusion protein of the disclosure (e.g., comprising an anti-CD8 antigen binding domain of the disclosure and a mutant IL-2 polypeptide) compared to the activation of Treg cells by wild-type IL-2, and by comparing the activation of NK cells (expressing IL-2Rbg) by a fusion protein of the disclosure (e.g., comprising an anti-CD8 antigen binding domain of the disclosure and a mutant IL-2 polypeptide) compared to the activation of NK cells by wild-type IL-2, or an IL-2 polypeptide with wild-type-like binding to IL-2Rb and IL2Rg but without binding to IL-2Ra.

[0021] In some embodiments, the binding affinity of a mutant IL-2 polypeptide with reduced or no binding affinity to IL-2Rα to IL-2Rβ or IL-2Rγ is measured by comparing the activation of cells expressing IL-2Rβ and IL-2Rγ by a fusion protein of the present disclosure (e.g., comprising an anti-CD8 antigen binding domain of the present disclosure and a mutant IL-2 polypeptide) compared to the activation of cells expressing IL-2Rβ or IL-2Rγ by wild-type IL-2 or an IL-2 polypeptide with wild-type-like binding to IL-2Rb and IL2Rg but not binding to IL-2Ra. For example, a mutant IL-2 polypeptide (or a fusion protein comprising the same) with reduced or no binding to IL-2Rα can be further mutated as necessary and tested for activation of cells expressing IL-2Rα / β / γ, e.g., Treg cells or IL-2bg, i.e., NK cells. Because mutant IL-2 polypeptides do not bind to IL-2Rα, the ability to activate cells expressing IL-2Rα / β / γ or the efficacy of activation of such cells can be used as an assay for binding of mutant IL-2 polypeptides or fusion proteins to IL-2Rβ / γ.

[0022] In some embodiments, the fusion protein activates CD8+ T cells 10-fold or more potently, or 50-fold or more potently, as compared to activating NK cells. In some embodiments, the mutant IL-2 polypeptide comprises the sequence of SEQ ID NO: 1 with one or more, or two or more amino acid substitutions relative to SEQ ID NO: 1, which substitutions are at positions in SEQ ID NO: 1 selected from the group consisting of Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130. In some embodiments, the mutant IL-2 polypeptide comprises an F42A or F42K amino acid substitution relative to SEQ ID NO: 1. In some embodiments, the mutant IL-2 polypeptide further comprises a R38A, R38D, R38E, E62Q, E68A, E68Q, E68K, or E68R amino acid substitution relative to SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide further comprises an H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K or S127Q amino acid substitution relative to SEQ ID NO:1. In some embodiments, the mutant IL-2 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-88. In some embodiments, the mutant IL-2 polypeptide comprises the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO: 1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A and N88S; R38E, F42A and N88A; R38E, F42A and V91E; R38E, F42A and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S;R38D, F42A and N88S;R38D, F42A and N88A;R38D, F42A and V91E;R38D, F42A and D84H;H16D, R38D and F42A;H16E, R38D and F42A;R38D, F42A and Q126S;R38A, F42K and N88S;R38A, F42K and N88A;R38A, F42K and V91E;R38A, F42K and D84H;H16D, R38A and F42K;H16E, R38A and F42K;R38A, F42K and Q126S;F42A, E 62Q and N88S;F42A, E62Q and N88A;F42A, E62Q and V91E;F42A, E62Q and D84H;H16D, F42A and E62Q;H16E, F42A and E62Q;F42A, E62Q and Q126S;R38E, F42A and C125A;R38D, F42A and C125A;F42A, E62Q and C125A;R38A, F42K and C125A;R38E, F42A, N88S and C125A;R38E, F42A, N88A and C125A;R38E, F42A, V91E and and C125A;R38E, F42A, D84H and C125A;H16D, R38E, F42A and C125A;H16E, R38E, F42A and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S and C125A;R38D, F42A, N88A and C125A;R38D, F42A, V91E and C125A;R38D, F42A, D84H and C125A;H16D, R38D, F42A and C125A;H16E, R38D, F42A and C125A;R38D, F42 A, C125A and Q126S;R38A, F42K, N88S and C125A;R38A, F42K, N88A and C125A;R38A, F42K, V91E and C125A;R38A, F42K, D84H and C125A;H16D, R38A, F42K and C125A;H16E, R38A, F42K and C125A;R38A, F42K, C125A and Q126S;F42A, E62Q, N88S and C125A;F42A, E62Q, N88A and C125A;F42A, E62Q, V91E and C125A;and F42A, E62Q, C125A and Q126S. In some embodiments, the variant IL-2 polypeptide is any of the variants described herein, including IL-2m1, IL-2m2, IL-2m3, IL-2m4, IL-2m4.9, IL-2m4.10, IL-2m4.11, IL-2m4.12, IL-2m4.13, IL-2m4.14, IL-2m4.15, IL-2m4.16, IL-2m4.17, IL-2m4.2, IL-2m4.1, IL-2m4.6, IL-2m4.18, IL-2m4.4, IL-2m4.19, IL-2m4.5, IL-2m4. 20, IL-2m4.3, IL-2m4.21, IL-2m4.22, IL-2m4.23, IL-2m4.24, IL-2m5, IL-2m6, IL-2m7, IL-2m8, IL-2m9, IL-2m10, IL-2m10.1, IL-2m10.2, IL-2m10.3, IL-2m10.4, IL-2m10.5, IL-2m10.6, IL-2m10.7, IL-2m10.8, IL-2m10.9, IL-2m10.10, IL-2m10.11. In some embodiments, the fusion protein binds human CD8, and binding of the fusion protein to CD8 does not block the interaction of CD8 with MHC class I. In some embodiments, the mutant IL-2 polypeptide further comprises the amino acid mutation C125A compared to SEQ ID NO: 1. In some embodiments, the first and second Fc domains comprise the following Fc mutations according to EU numbering: L234A, L235A, G237A and K322A. In some embodiments, the first Fc domain comprises the following amino acid substitutions: Y349C and T366W and the second Fc domain comprises the following amino acid substitutions according to EU numbering: S354C, T366S, L368A and Y407V;or the second Fc domain comprises the following amino acid substitutions: Y349C and T366W, and the first Fc domain comprises the following amino acid substitutions according to EU numbering: S354C, T366S, L368A, and Y407V. In some embodiments, the fusion protein has or exhibits one or more of the following properties: binding to human CD8, and the binding of the fusion protein to CD8 does not block the interaction of CD8 with MHC class I; and the fusion protein activates CD8+ T cells with 10-fold or greater potency compared to activation of NK cells. In some embodiments, the potency of activation of CD8+ T cells and NK cells is measured by the EC50 of cell activation assessed by cell proliferation (e.g., Ki67 assay). In some embodiments, the potency of activation of CD8+ T cells and NK cells is measured by the EC50 of cell activation assessed by STAT5 activity (e.g., pSTAT5 assay).;

[0023] Other aspects of the disclosure relate to an isolated polynucleotide(s) encoding a mutant IL-2 polypeptide or fusion protein according to any one of the above embodiments. Other aspects of the disclosure relate to a vector(s) encoding a mutant IL-2 polypeptide, fusion protein or isolated polynucleotide according to any one of the above embodiments. In some embodiments, the vector(s) is an expression vector. Other aspects of the disclosure relate to a host cell (e.g., an isolated and / or recombinant host cell) comprising a polynucleotide and / or vector according to any one of the above embodiments. Other aspects of the disclosure relate to a pharmaceutical composition comprising a fusion protein according to any one of the above embodiments and a pharma- ceutically acceptable carrier. Other aspects of the disclosure relate to the use of a fusion protein or pharmaceutical composition according to any one of the above embodiments as a medicament. Other aspects of the disclosure relate to the use of a fusion protein or pharmaceutical composition according to any one of the above embodiments in the manufacture of a medicament. Another aspect of the present disclosure relates to the use of a fusion protein or pharmaceutical composition according to any one of the above embodiments in a method for treating cancer or a chronic infection, the method comprising administering to a patient in need thereof an effective amount of the fusion protein or pharmaceutical composition. Another aspect of the present disclosure relates to the use of a fusion protein or pharmaceutical composition according to any one of the above embodiments in a method for treating cancer, the method comprising administering to a patient in need thereof an effective amount of the fusion protein or pharmaceutical composition in combination with a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). Another aspect of the present disclosure relates to the use of a fusion protein or pharmaceutical composition according to any one of the above embodiments in the manufacture of a medicament for treating cancer or a chronic infection. Another aspect of the present disclosure relates to a method for treating cancer or a chronic infection, the method comprising administering to a patient in need thereof an effective amount of a fusion protein or pharmaceutical composition according to any one of the above embodiments.Another aspect of the present disclosure relates to a method of treating cancer, comprising administering to a patient in need thereof an effective amount of a fusion protein or pharmaceutical composition according to any one of the above embodiments in combination with a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). In some embodiments according to any of the embodiments described herein, the ICI is an inhibitor of PD-1, PD-L1, or CTLA-4.

[0024] In some embodiments, targeted IL-2 fusion proteins of the present disclosure containing an antigen binding protein activate antigen-expressing IL-2Rβ+ cells, e.g., CD8+ T cells, at least 10-fold, 50-fold, 100-fold, or at least 200-fold over non-antigen-expressing IL-2Rβ+ cells, e.g., NK cells. In some embodiments, fusion proteins of the present disclosure activate antigen-expressing IL-2Rβ+ cells more than 50-fold, 100-fold, or at least 200-fold, for example, compared to a fusion protein comprising the IL-2 variant polypeptide and a control antibody that does not bind any antigen expressed on the cells. The cell activation by IL-2 fusion proteins is determined in an in vitro assay by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in the cells following treatment with the IL-2 fusion protein.

[0025] In summary, the present disclosure achieves a reduction in the pleiotropic effects of IL-2 on any immune cell expressing the IL-2R complex to a subset of effects by reducing the effects of IL-2 to a specific immune cell subset of interest, such as CD8+ T cells. Such focus aims to direct the action of IL-2 polypeptides when administered as a therapeutic to T cell subsets containing tumor antigen-specific CD8+ T cells or viral antigen-specific CD8+ T cells, thus reducing the toxicity of IL-2 polypeptides by sparing 1) T cells that may not contribute to efficacy; or 2) innate lymphocytes that express the receptor for IL-2, are distributed throughout the body, and may contribute to toxicity; and 3) other immune cells that may act as a sink for IL-2 or negatively contribute to efficacy. The present invention provides, for example, the following items. (Item 1) A fusion protein comprising two parts: i) a first portion comprises an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer, where VH is the variable heavy chain and CH2-CH3 is the Fc domain, an antibody light chain VL-CL, where VL is the variable light chain and CL is the constant light chain, and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second portion comprises an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; wherein both the first portion and the second portion bind to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1; Fusion proteins. (Item 2) A fusion protein comprising two parts: i) a polypeptide comprising an antibody hinge-CH2-CH3 monomer, where the first portion is an antibody hinge-CH2-CH3 monomer, where CH2-CH3 is an Fc domain, and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1; Fusion proteins. (Item 3) A fusion protein comprising two parts: i) a polypeptide comprising an antibody hinge-CH2-CH3 monomer, where the first portion is an antibody hinge-CH2-CH3 monomer, where CH2-CH3 is an Fc domain, and a mutant IL-2 polypeptide, wherein the C-terminus of the mutant IL-2 polypeptide is fused to the N-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1; Fusion proteins. (Item 4) A fusion protein comprising two parts: i) the first portion comprises an antigen-binding domain that binds to human CD8α or human CD8β; ii) the second portion comprises a mutant IL-2 polypeptide; the second portion is linked to the first portion via a linker; Fusion proteins. (Item 5) 5. The fusion protein of item 4, wherein the first portion comprises: (a) an antibody or an antigen-binding fragment thereof comprising one or two heavy chain polypeptides and one or two light chain polypeptides; (b) a single chain antibody or a single chain variable fragment (scFv); or (c) a VHH antibody. (Item 6) 6. The fusion protein of any one of items 1 to 5, which activates CD8+ T cells with 10-fold or greater potency compared to activation of NK cells. (Item 7) 7. The fusion protein of item 6, which activates CD8+ T cells with 50-fold or greater potency compared to activation of NK cells. (Item 8) 8. The fusion protein of any one of items 1 to 7, wherein the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having the amino acid sequence of SEQ ID NO:2 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. (Item 9) The fusion protein of item 8, wherein the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rβ polypeptide having the amino acid sequence of SEQ ID NO:3 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. (Item 10) 10. The fusion protein of claim 8 or 9, wherein the mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rγ polypeptide having the amino acid sequence of SEQ ID NO:4 compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence of SEQ ID NO:1. (Item 11) The mutant IL-2 polypeptide comprises the sequence of SEQ ID NO:1 having one or more or two or more amino acid substitutions relative to SEQ ID NO:1, 11. The fusion protein of any one of items 1 to 10, wherein the or more than one or two or more substitutions comprise a substitution at a position of SEQ ID NO: 1 selected from the group consisting of Q11, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129 and S130. (Item 12) 12. The fusion protein of item 11, wherein the one or more or two or more substitutions comprise an F42A or F42K amino acid substitution relative to SEQ ID NO:1. (Item 13) 13. The fusion protein of claim 11 or 12, wherein the one or more or two or more substitutions further comprise a R38A, R38D, R38E, E62Q, E68A, E68Q, E68K or E68R amino acid substitution relative to SEQ ID NO:1. (Item 14) 14. The fusion protein of any one of items 11 to 13, wherein the one or more or two or more substitutions further comprise a H16E, H16D, D20N, M23A, M23R, M23K, S87K, S87A, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K or S127Q amino acid substitution relative to SEQ ID NO:1. (Item 15) 15. The fusion protein according to any one of items 11 to 14, wherein the one or more or two or more substitutions further comprise the amino acid mutation C125A compared to SEQ ID NO:1. (Item 16) 11. The fusion protein according to any one of items 1 to 10, wherein the mutant IL-2 polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18 to 88. (Item 17) The mutant IL-2 polypeptide has the following set of amino acid substitutions (relative to the sequence of SEQ ID NO:1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A and N88S; R38E, F42A and N88A; R38E, F42A and V91E; R38E, F42A and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q1 26S;R38D, F42A and N88S;R38D, F42A and N88A;R38D, F42A and V91E;R38D, F42A and D84H;H16D, R38D and F42A;H16E, R38D and F42A;R38D, F42A and Q126S;R38A, F42K and N88S;R38A, F42K and N88A;R38A, F42K and V91E;R38A, F42K and D84H;H16D, R38A and F42K;H 16E, R38A and F42K;R38A, F42K and Q126S;F42A, E62Q and N88S;F42A, E62Q and N88A;F42A, E62Q and V91E;F42A, E62Q and D84H;H16D, F42A and E62Q;H16E, F42A and E62Q;F42A, E62Q and Q126S;R38E, F42A and C125A;R38D, F42A, and C125A;F42A, E62Q and C125A;R 38A, F42K and C125A;R38E, F42A, N88S and C125A;R38E, F42A, N88A and C125A;R38E, F42A, V91E and C125A;R38E, F42A, D84H and C125A;H16D, R38E, F42A and C125A;H16E, R38E, F42A and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S and C125A;R38D, F42 A, N88A and C125A;R38D, F42A, V91E and C125A;R38D, F42A, D84H and C125A;H16D, R38D, F42A and C125A;H16E, R38D, F42A and C125A;R38D, F42A, C125A and Q126S;R38A, F42K, N88S and C125A;R38A, F 42K, N88A and C125A;R38A, F42K, V91E and C125A;R38A, F42K, D84H and C125A;H16D, R38A, F42K and C125A;H16E, R38A, F42K and C125A;R38A, F42K, C125A and Q126S;F42A, E62Q, N88S and C125A;F42A, E62Q, N88A and C125A;F42A, E62Q, V91E and C125A;F42A, E62Q and D84H and C125A;H16D, F42A and E62Q and C125A;H16E, F42A, E62Q and C125A;F42A, E62Q, C125A and Q126S;F42A, N88S and C125A;F42A 11. The fusion protein of any one of items 1 to 10, comprising the amino acid sequence of SEQ ID NO: 1, having one of the following: N88A and C125A; F42A, V91E and C125A; F42A, D84H and C125A; H16D, F42A and C125A; H16E, F42A and C125A; and F42A, C125A and Q126S. (Item 18) 18. The fusion protein according to any one of items 1 to 17, wherein the fusion protein binds to human CD8 and the binding of the fusion protein to CD8 does not block the interaction of CD8 with MHC class I. (Item 19) 19. The fusion protein of any one of items 1 to 18, wherein the first and second Fc domains comprise the following Fc mutations according to EU numbering: L234A, L235A, G237A and K322A. (Item 20) (a) the first Fc domain comprises the following amino acid substitutions: Y349C and T366W and the second Fc domain comprises the following amino acid substitutions: S354C, T366S, L368A and Y407V, according to EU numbering; or (b) the second Fc domain comprises the following amino acid substitutions: Y349C and T366W, and the first Fc domain comprises the following amino acid substitutions: S354C, T366S, L368A, and Y407V, according to EU numbering; 20. The fusion protein of any one of items 1 to 19. (Item 21) (a) the property that the fusion protein binds to human CD8 and that the binding of the fusion protein to CD8 does not block the interaction of CD8 with MHC class I; and (b) activating CD8+ T cells with 10-fold or greater potency compared to activating NK cells; 21. The fusion protein of any one of items 1 to 20, having one or more of: (Item 22) 22. The fusion protein of any one of items 6, 7 and 21, wherein the potency of activating CD8+ T cells and NK cells is measured by EC50 of cell activation assessed by cell proliferation. (Item 23) 23. One or more isolated polynucleotides encoding the mutant IL-2 polypeptide or fusion protein of any one of items 1 to 22. (Item 24) 24. One or more vectors, particularly expression vectors, comprising the polynucleotide according to item 23. (Item 25) A host cell comprising the polynucleotide of item 23. (Item 26) 23. A pharmaceutical composition comprising the fusion protein according to any one of items 1 to 22 and a pharma- ceutically acceptable carrier. (Item 27) 27. The fusion protein according to any one of items 1 to 22 or the composition according to item 26 for use as a medicament. (Item 28) 27. A method for treating cancer or a chronic infection, comprising administering to a patient an effective amount of the fusion protein of any one of items 1 to 22 or the composition of item 26. (Item 29) A method for treating cancer, comprising administering to a patient an effective amount of the fusion protein of any one of items 1 to 22 or the composition of item 26 in combination with a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). (Item 30) 30. The method of item 29, wherein the ICI is an inhibitor of PD-1, PD-L1 or CTLA-4. [Brief description of the drawings]

[0026] [Figure 1] 1A-1D show the amino acid sequences of mature IL-2 (FIG. 1A; SEQ ID NO:1), IL-2Rα (FIG. 1B; SEQ ID NO:2), IL-2Rβ (FIG. 1C; SEQ ID NO:3) and IL-2Rγ (FIG. 1D; SEQ ID NO:4) polypeptides.

[0027] [Diagram 2] 2 shows the amino acid sequence of the wild-type mature IL-2 polypeptide (SEQ ID NO:1). "X" indicates an amino acid in the sequence of the wild-type IL-2 polypeptide that is replaced with another amino acid to generate the mature IL-2 polypeptide of the present disclosure.

[0028] [Diagram 3] FIG. 3 shows the general mechanism of how targeted fusions of mutant IL-2 polypeptides to CD8 or PD1 antigen binding molecules, and non-targeted fusions to mutant IL-2 polypeptides, act to stimulate cells that do or do not express the CD8 or PD1 antigens.

[0029] [Figure 4]FIG. 4 depicts three different fusion protein formats (formats A, B and C) according to some embodiments.

[0030] [Diagram 5] FIG. 5 shows activation of STAT5 in different mouse spleen cell subsets stimulated with therapeutic human IL-2 (left) and with an IL-2 variant that has no binding to IL-2Rα and wild-type binding to IL-2Rβ and IL-2Rγ (right). STAT5 activation in spleen subsets was measured by flow cytometry. STAT5 activation in spleen cell subsets stimulated with IL-2 is shown on the left. STAT5 activation in spleen cell subsets stimulated with xHA-IL-2v, a fusion of a previously published IL-2 variant that does not bind IL-2Ra (IL-2v; see Klein et al, Oncoimmunol. 2017; 6(3); e1277306) with a control antibody (xHA), is shown on the right. NK cells were found to be more sensitive to IL-2 and IL-2 variants with reduced binding to CD25 / IL2Rα than CD8 T cells.

[0031] [Figure 6]Figures 6A and 6B show NK cell-induced toxicity of IL-2 variants with reduced binding to CD25 in mice. 8-10 week old B6 mice were injected subcutaneously with a single dose of the indicated compound and their body weights were recorded daily. Figure 6A shows body weight records for mice treated with xHA-IL-2v administered in combination with 2.5 mg / kg of anti-PD1 (xPD1). Figure 6B shows body weight records for mice treated with TAg-IL-2v, the same IL-2v fused to an antibody targeting an antigen expressed in tumors (tumor antigen / TAg). TAg-IL-2v was administered alone at 5 mg / kg. NK cells were depleted with 200 mg / mouse ip of anti-NK1.1 antibody (PK136 clone). Depleting antibody was injected 2 days before and 1 day after TAg-IL-2v administration to maintain depletion. NK cells induced toxicity that was manifested as weight loss in mice treated with an IL-2 variant that had reduced binding to CD25 / IL2Ra.

[0032] [Figure 7] Figure 7 shows the determination of binding of anti-mouse CD8 antibodies to CD8+ T cells. Fresh splenocytes were incubated with the indicated antibodies for 2 hours at 4°C. Cells were then stained with antibodies against CD3, CD4, CD8 and anti-hFc. Anti-hFc was used to measure binding of CD8-IL2 fusions containing hFc. Cells were washed and analyzed by flow cytometry. The mean fluorescence intensity (MFI) of staining with anti-hFc is used to indicate binding. xmCD8ab2 (published clone YTS156.7.7) had a higher affinity than xmCD8ab1 (published clone 2.43). xCD8ab2.1 is a lower affinity variant of xCD8ab2, generated by introducing two mutations into xCD8ab2.

[0033] [Figure 8]Figure 8 shows the MHC blocking status for anti-mouse CD8 antibodies. CD8+ T cells were purified from splenocytes from OT-I mice and co-cultured with EL-4-OVA expressing line (E.G7-OVA, CRL-2113; ATCC) at 100,000 cells each for 24 hours. Cells were analyzed by cell surface staining and flow cytometry for upregulation of activation markers such as CD25 and CD69. Both xCD8ab1 and xCD8ab2 blocked T cell activation as measured by % of cells expressing CD25. xCD8ab2 blocked T cell activation more potently, correlating with its higher binding affinity to CD8.

[0034] [Figure 9] Figure 9 shows selective targeting of CD8 T cells by human IL-2 muteins fused to CD8 antibodies over other immune cells expressing IL-2R. IL-2 muteins were fused to a previously published anti-mouse CD8 antibody, xmCD8ab1 (2.43 clone), in format B (schematically depicted on the left). STAT5 activation in mouse splenocytes was measured by flow cytometry. IL-2 mutein variants fused to CD8 antibodies selectively targeted CD8+ T cells (upper left graph) over other immune cells expressing IL-2R, such as NK cells identified as CD3-CD49b+ (upper right), CD4+CD25-Tconv cells (lower left) and CD4+CD25+Treg cells (lower right).

[0035] [Figure 10]FIG. 10 shows the efficacy of a single dose of CD8-IL-2 versus a single dose of TAg-IL-2v in combination with anti-PD1 in a B16 cold tumor model. C57BL6 mice were implanted subcutaneously with 5×105 cells (100 μL) of cultured B16.F10 cells (ATCC, CRL-6475) in the upper rear of the hind limb. Tumor volumes were measured (TV=width×width×length×0.5) until the tumor volume reached 60-120 mm3 approximately 8 days after implantation. Each mouse was weighed and administered the indicated compound (10 mice / group) subcutaneously under the nape of the neck: PBS (upper left), xmCD8ab1-IL2m10 at 1 mg / kg (upper center), xmCD8ab2-IL2m10 at 1 mg / kg (upper right), xPD1 at 5 mg / kg (lower left), TAg-IL-2v at 1 mg / kg (lower center), or TAg-IL-2v at 3 mg / kg (lower right). Tumor volumes and body weights were measured every 3-4 days until the end of the study (30-40 days after first dose) or until maximum tumor volume (2000 mm3) was reached. Complete regression (CR) of tumors and partial regression or slower growth (PR) of tumors defined by <100 mm3 by day 14 post-dose are indicated where applicable. All mice except those in the PBS and xPD1 groups were co-administered with anti-PD1. xmCD8-IL2m10 performed better than TAg-IL-2v in combination with anti-PD1 in the B16 cold tumor model.

[0036] [Figure 11]Figures 11A and 11B show induction of CD8 T cell accumulation in the blood and tumors of B16 tumor-bearing mice treated with a single dose of CD8-IL-2. B6 mice were injected with B16 tumor cells and tumors were allowed to grow to 200-250 mm3, after which the mice were administered the indicated IL-2 fusions at 1 mg / kg along with xPD1 at 5 mg / kg. Cells were harvested from tumors and blood and profiled by flow cytometry to detect CD8+ T cells and NK cells (NK1.1+CD3-) as indicated. Figure 11A shows immune cell counts in the blood and Figure 11B shows immune cell counts in the tumor. More CD8+ T cells were observed in the tumor with xmCD8-IL2m10 than with xHA-IL-2v.

[0037] [Figure 12] Figures 12A-12C show the performance of a single dose of CD8-IL-2 and a single dose of TAg-IL-2v in the CT26 tumor model. BALB / c female mice were implanted subcutaneously with 2 x 105 cells (100 μL) of cultured CT26.wt cells (ATCC, CRL-2638) in the upper rear of the hind limb. Tumor volumes were measured (TV = width x width x length x 0.5) until the tumor volume reached 60-120 mm3 approximately 8 days after implantation. Each mouse was then weighed and administered the indicated compound subcutaneously (9 mice per group): PBS (Figure 12A), TAg-IL-2v at 2 mg / kg (Figure 12B), or xmCD8ab2-IL2m4 at 0.3 mg / kg (Figure 12C). Tumor volumes and body weights were measured every 3-4 days until the end of the study (30 days after the first dose) or until maximum tumor volume (2000 mm3) was reached. Complete regression (CR) of tumors is indicated where applicable. xmCD8-IL2m4 performed better than TAg-IL-2v in the CT26 tumor model.

[0038] [Figure 13]Figure 13 shows the effect of CD8 antibody affinity on fusion potency in vitro. Cells were treated with the IL-2 mutein IL-2m4 fused to xmCD8ab2 or its lower affinity variant xmCD8ab2.1 in format C. STAT5 activation in CD8+ T cells (left) and NK cells (right) was measured by flow cytometry. xmCD8ab2.1-IL2m4 had lower potency and lower selectivity for CD8 T cells over NK cells compared to xmCD8ab2-IL-2m4.

[0039] [Figure 14] FIG. 14 shows the in vivo expansion of CD8+ T cells treated with xmCD8ab2-IL2m4 and xmCD8ab2.1-IL2m4. Naive B6 mice were treated with the indicated compounds at 1 mg / kg and bled 5 days after dosing. Cells were stained with lineage markers to identify CD8+ T cells and NK cells and profiled by flow cytometry. Both xmCD8ab2-IL2m4 and xmCD8ab2.1-IL2m4 expanded CD8 T cells in vivo. Both fusions induced a higher expansion of CD8 T cells than NK cells in vivo.

[0040] [Figure 15]Figures 15A and 15B show the characterization in a STAT5 assay of xmCD8-IL-2 mutant protein, which has no binding to IL-2Rα and has one additional mutation, fused to a high affinity CD8 antibody. Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins and then stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for intracellular phospho-STAT5 (pSTAT5). Cells were analyzed by flow cytometry. Data show the mean fluorescence intensity (MFI) for STAT5 in the indicated cell subsets. Figure 15A shows the activation of STAT5 in CD8+ T cells, while Figure 15B shows the activation of STAT5 in NK cells (defined as CD3-CD49b+). Certain IL-2 mutations reduced binding of IL-2 mutant proteins fused to anti-CD8 antibodies to IL2Rβ / γ-expressing cells while retaining higher potency against CD8+ T cells than other IL2Rβ / γ-expressing cells that do not express CD8.

[0041] [Figure 16] Figures 16A and 16B show the characterization in a STAT5 assay of xmCD8-IL-2 muteins, which have no binding to IL-2Rα and have one additional mutation, fused to a low affinity CD8 antibody variant. Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins and then stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for intracellular STAT5. Cells were analyzed by flow cytometry. Data show the mean fluorescence intensity (MFI) for STAT5 in the indicated cell subsets. Figure 16A shows the activation of STAT5 in CD8+ T cells, while Figure 16B shows the activation of STAT5 in NK cells. Certain IL-2 mutations reduced the binding of IL-2 muteins fused to low affinity anti-CD8 antibodies to IL2Rβ / γ expressing cells while maintaining a higher potency for CD8+ T cells than other IL2Rβ / γ expressing cells that do not express CD8.

[0042] [Figure 17] Figures 17A and 17B show the characterization of xmCD8-IL-2 mutant proteins, which have no binding to IL-2Rα and have one additional mutation, fused to a high affinity CD8 antibody, in an assay that detects the expression of a cell proliferation marker (Ki67). Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins and then stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for intracellular markers of proliferation, Ki67, downstream signaling events from IL2Rβ / γ and STAT5. Data show the % of cells in the indicated cell subsets positive for the proliferation marker Ki67. Figure 17A shows the percentage of Ki67 positive CD8+ T cells, while Figure 17B shows the percentage of Ki67 positive NK cells.

[0043] [Figure 18] Figures 18A and 18B show the characterization in the Ki67 assay of xmCD8-IL-2 mutant proteins, which have no binding to IL-2Rα and one additional mutation, fused to a low affinity CD8 antibody. Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins and then stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for Ki67. Data show the % of cells in the indicated cell subsets positive for the proliferation marker Ki67. Figure 18A shows the percentage of Ki67 positive CD8+ T cells, while Figure 18B shows the percentage of Ki67 positive NK cells.

[0044] [Figure 19]FIG. 19 depicts an overview of the potency for CD8 T cells and NK cells for representative molecules and their selectivity for CD8 T cells over NK cells. CD8 and NK cell activation for each molecule are shown on the same graph. The difference in potency for CD8 cells versus NK cells is indicated by a double-headed arrow. The graph summarizes the characterization data for generated CD8-IL2 fusions with various selectivities for CD8+ T cells over NK cells. xmCD8ab2-IL2m4 (top left) and xmCD8ab2-IL2m4.2 (top right) had the highest selectivity (>1000-fold), followed by xmCD8ab2.1-IL2m4 (~50-100-fold, bottom left), and xmCD8ab2.1-IL2m4.1 (~10-fold; bottom right) had the lowest selectivity.

[0045] [Figure 20] FIG. 20 depicts the characteristics of the CD8-IL-2 fusion with the highest efficacy. Four representative CD8-IL2 fusions (as indicated) with varying degrees of selectivity for CD8 T cells over NK cells were tested in the B16 tumor model. The number of mice with complete regression (CR) out of all mice is shown in each panel. All mice were administered 1 mg / kg of the indicated fusion along with 5 mg / kg of anti-PD1. Dosing above 1 mg / kg may induce NK cell activation due to binding of IL-2 mutant proteins to IL-2Rβγ on NK cells, thereby inducing weight loss and toxicity. CD8-IL-2 performed better than TAg-IL-2v at lower doses. The CD8-IL2 fusion with the lowest selectivity for CD8 T cells had the least efficacy in the B16 model, approaching that observed for TAg-IL-2v in FIG. 10, where only 1 in 10 mice showed complete tumor regression. Highest efficacy, therapeutic index, and >40% tumor-free mice required >10-fold selectivity.

[0046] [Figure 21]Figure 21 shows the expansion proliferation of tumor antigen-specific CD8+ T cells and total CD8+ T cells and NK cells by treatment with CD8-IL-2. B6 mice were injected with B16 tumor cells and tumors were allowed to grow to 200-250 mm3, after which the mice were administered the indicated IL-2 fusions at 1 mg / kg together with xPD1 at 5 mg / kg. Tumors were removed 5 days after administration, digested to single cells, and profiled by flow cytometry to detect CD8+ T cells and NK cells (NK1.1+CD3-). Cells were also stained with p15E tetramer (TB-M507-2, MBL) according to the manufacturer's protocol to detect T cells that recognize the p15E tumor antigen. Data are expressed as cell counts per 106 cells isolated from each tumor. Both xmCD8ab2-IL2m4.2 and xmCD8ab2.1-IL2m4 induced an approximately 15-fold expansion of total intratumoral CD8+ T cells and a 5- to 17-fold expansion of p15E tumor antigen-specific T cells.

[0047] [Figure 22] Figure 22 shows the potency of bivalent low affinity CD8 antibody IL-2 fusions and monovalent high affinity CD8 antibody IL-2 fusions. Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins for 30 minutes in RPMI medium, after which the cells were stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for intracellular phospho-STAT5. The bivalent low affinity fusions had similar potency as that of the high affinity monovalent fusions, as measured by the percentage of cells positive for pSTAT5. IL-2m4.2 fusions fused to the high affinity xmCD8ab2 antibody (in format C) or to the bivalent xmCD8ab2.1 antibody (in format A) had similar potency on CD8+ T cells and much greater potency than the monovalent xmCD8ab2.1-IL-2m4 (format C) fusion.

[0048] [Figure 23]Figure 23 shows the efficacy of bivalent C-terminal format (format A) fusions in the B16 tumor model. Mice were administered PBS as a control or 1 mg / kg of the indicated fusions together with 5 mg / kg of anti-PD1 (9 mice per group). The bivalent C-terminal format (format A) was also highly effective. IL-2m4.2 fusions fused to the high affinity xmCD8ab2 antibody in format C (Figure 20) or to the bivalent xmCD8ab2.1 antibody in format A (Figure 23) had similar in vivo efficacy.

[0049] [Figure 24] Figure 24 shows blocking of CD8 T cell activation by CD8 antibodies. CD8+ T cells were purified from splenocytes from OT-I mice and co-cultured with EL-4-OVA line (ATCC) at 100,000 cells each for 24 hours. Cells were analyzed by cell surface staining and flow cytometry for upregulation of activation markers such as CD25 and CD69. Certain CD8 antibodies did not block CD8 T cell activation. The xmCD8ab3 antibody (comprising a VH domain comprising the sequence of SEQ ID NO: 16 and a VL domain comprising the sequence of SEQ ID NO: 17) did not block CD8 T cell activation even at a concentration of 200 nM. The xmCD8ab3 antibody was in a bivalent format.

[0050] [Diagram 25] Figure 25 shows a comparison of the in vitro potency of xmCD8ab2 and xmCD8ab3 fusions as indicated. Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins for 30 minutes in RPMI medium, after which cells were stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b) and for intracellular pSTAT5. Both xmCD8ab2-IL2m4.2 and xmCD8ab3-IL2m4.2 showed similar activity and greater potency than TAg-IL-2v on CD8+ T cells in vitro.

[0051] [Figure 26] Figures 26A and 26B show the in vivo efficacy of non-MHC-blocked anti-CD8 antibodies fused to IL-2 muteins in a B16 tumor model. Mice were administered PBS or 0.3 mg / kg (Figure 26A) or 1 mg / kg (Figure 26B) of the indicated fusions together with 5 mg / kg anti-PD1. IL-2m4.2 fusions fused to non-MHC-blocked xmCD8ab3 antibodies in format C (Figure 26B) were much more effective than IL-2m4.2 fusions fused to MHC-blocked xmCD8ab2 antibodies in format C (Figure 26A).

[0052] [Figure 27] Figure 27 shows fusions of IL-2 mutant proteins preferentially target PD1+ T cells over PD1- T cells. B16 tumors measuring 300-600 mm3 in size were removed from mice and digested to single cells. CD45+ cells were purified (Miltenyi LS columns, following manufacturer's protocol) and stimulated with the indicated fusion proteins for 30 min. Cells were stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b, and PD1) and for intracellular phospho-STAT5. Fusions of IL-2 mutant protein IL2m10 and anti-PD1 antibody preferentially targeted PD1+ T cells over PD1- T cells, but targeted both CD8+PD1+ T cells and CD4+CD25+PD1+ Treg cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] definition "Immune cells", as used herein, are cells of the immune system that react against organisms or other entities that are considered foreign to the host's immune system. They protect the host from foreign pathogens, organisms, and diseases. Immune cells, also called white blood cells, are involved in both innate and adaptive immune responses to fight pathogens. Innate immune responses occur immediately upon exposure to a pathogen without further antigen stimulation or learning processes. Adaptive immune responses require an initial antigen stimulation followed by memory formation, resulting in enhanced responsiveness during subsequent encounters with the same pathogen. Innate immune cells include, but are not limited to, monocytes, macrophages, dendritic cells, innate lymphoid cells (ILCs), such as natural killer (NK) cells, neutrophils, megakaryocytes, eosinophils, and basophils. Adaptive immune cells include B and T lymphocytes / cells. T cell subsets include, but are not limited to, alpha beta CD4+T (naive CD4+, memory CD4+, effector memory CD4+, effector CD4+, regulatory CD4+), and alpha beta CD8+T (naive CD8+, memory CD8+, effector memory CD8+, effector CD8+). B cell subsets include, but are not limited to, naive B, memory B, and plasma cells. NK T and T gamma delta (Tγδ) cells display characteristics of both innate and adaptive lymphocytes.

[0054] "T cells" or "T lymphocytes" are immune cells that play a key role in orchestrating immune responses in health and disease. There are two major T cell subsets with distinct functions and properties: T cells that express the CD8 antigen (CD8 + T cells) are cytotoxic or killer T cells that can lyse target cells using cytotoxic proteins such as granzymes and perforin, whereas T cells that express the CD4 antigen (CD4 + T cells) are CD8 + They are helper T cells that can regulate the function of many other immune cell types, including those of T cells, B cells, and macrophages. In addition, CD4 +T cells are further subdivided into several subsets, such as regulatory T (Treg) cells, which can suppress immune responses, and helper T cell 1 (Th1), helper T cell 2 (Th2) and helper T cell 17 (Th17), which regulate different types of immune responses by secreting immunomodulatory proteins such as cytokines. T cells recognize their targets by alpha-beta T cell receptors that bind to unique antigen-specific motifs, and this recognition mechanism is generally required to trigger their cytotoxic and cytokine-secreting functions. "Innate lymphocytes" are CD8 T cells that express specific functions such as cytotoxic activity or secretion of Th1, Th2 and Th17 cytokines. + and CD4 + They can also show the characteristics of T cells. Some of these innate lymphocyte subsets include NK cells and ILC1, ILC2 and ILC3 cells; and innate-like T cells, such as Tγδ cells; and NK T cells. Typically, these cells can respond quickly to inflammatory stimuli from infected or injured tissue, such as immunoregulatory cytokines, but unlike alpha beta T cells, they can respond without the need to recognize antigen-specific patterns.

[0055] A "cytokine" is a form of immunomodulatory polypeptide that mediates crosstalk between progenitor / primary cells and target / effector cells. Cytokines can function as soluble forms or in association with the cell surface to bind to "cytokine receptors" on target immune cells and activate signaling. A "cytokine receptor," as used herein, is a cell surface polypeptide that activates intracellular signaling upon binding to a cytokine on the extracellular surface of the cell. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cytokines are produced by a wide range of cells, including immune cells, endothelial cells, fibroblasts, and stromal cells. A given cytokine may be produced by more than one cell type. Cytokines are pleiotropic; one cytokine can activate signaling pathways in multiple cells because receptors are expressed on multiple immune cell subsets. However, depending on the cell type, cytokine signaling events can result in different downstream cellular events, such as activation, proliferation, survival, apoptosis, effector functions, and secretion of other immunomodulatory proteins.

[0056] "Amino acid," as used herein, refers to naturally occurring carboxy α-amino acids, including alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y) and valine (val, V).

[0057] "Polypeptide" or "protein" as used herein refers to a molecule in which monomers (amino acids) are linked together in a linear chain by peptide bonds (also known as amide bonds). The term "polypeptide" refers to any chain of two or more amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "proteins", "amino acid chains", or any other term used to refer to a chain of two or more amino acids are included in the definition of "polypeptide", and the term "polypeptide" may be used in place of or synonymously with any of these terms. The term "polypeptide" is also intended to refer to the product of a polypeptide, which may be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. Polypeptides may be produced in any manner, including by chemical synthesis. Polypeptides may usually have a defined three-dimensional structure, but do not necessarily have such a structure. The polypeptides of the present disclosure may be of a size of about 3 or more, 5 or more, 10 or more, 20 or more, 25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. A polypeptide that has a defined three-dimensional structure is referred to as folded, and a polypeptide that does not have a defined three-dimensional structure, but rather adopts a number of different conformations, is referred to as unfolded. Polypeptides may further form multimers, such as dimers, trimers, and higher oligomers, i.e., consisting of more than one polypeptide molecule. The polypeptide molecules that form such dimers, trimers, etc. may be identical or non-identical. Thus, the corresponding higher order structures of such multimers are referred to as homo- or hetero-dimers, homo- or hetero-trimers, etc.The terms "polypeptide" and "protein" also refer to modified polypeptides / proteins that are subject to post-expression modifications, including, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modifications with non-naturally occurring amino acids.

[0058] "Residue" as used herein refers to the position in a protein and the identity of its associated amino acid. For example, Leu 234 (also called Leu234 or L234) is the residue at position 234 in human antibody IgG1.

[0059] "Wild-type" refers herein to an amino acid or nucleotide sequence found in nature, including allelic variations. A wild-type protein has an amino acid or nucleotide sequence that has not been intentionally modified.

[0060] "Substitution" or "mutation" refers to a change to a polypeptide backbone in which an amino acid naturally occurring in the wild-type sequence of the polypeptide is replaced with another amino acid that does not naturally occur at the same position in the polypeptide. Preferably, the mutation(s) are introduced to modify the activity of the polypeptide such that the affinity of the polypeptide for its receptor is altered, thereby making the polypeptide have an affinity and activity that differs from that of the wild-type cognate polypeptide. Mutations can also improve the biophysical properties of the polypeptide. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, and the like. It is believed that methods of modifying the side chain group of an amino acid by methods other than genetic engineering, such as chemical modification, can also be useful.

[0061] "Interleukin-2" or "IL-2", as used herein, refers to any native human IL-2, unless otherwise indicated. "IL-2" encompasses unprocessed IL-2 as well as "mature IL-2", a form of IL-2 that results from processing within a cell. The sequence of "mature IL-2" is depicted in FIG. 1A. One exemplary form of unprocessed human IL-2 consists of an additional N-terminal amino acid signal peptide attached to mature IL-2. "IL-2" includes, but is not limited to, naturally occurring variants of IL-2, such as alleles or splice variant(s). The amino acid sequence of an exemplary human IL-2 is described under UniProt P60568 (IL2_HUMAN).

[0062] "Affinity" or "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). Affinity is determined by the dissociation constant (K), which is the ratio of the dissociation rate constant to the association rate constant (koff and kon, respectively). D ) can be generally expressed by the rate constants (R=R, R=1, R=2, R=1). Thus, equivalent affinities may include different rate constants, provided that the ratio of rate constants remains the same. Affinity can be measured using common methods known in the art, such as enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., BIAcore), biolayer interference (BLI) (e.g., Octet) and other traditional binding assays (Heeley, Endocr Res 28, It can be measured by the method described above.

[0063] "Binding" or "specific binding", as used herein, refers to the ability of a polypeptide or antigen-binding molecule to selectively interact with the receptor of the polypeptide or target antigen, respectively, and this specific interaction can be distinguished from non-targeted or undesired or non-specific interactions. Examples of specific binding include, but are not limited to, the binding of IL-2 cytokine to its specific receptor (e.g., IL-2Rα, IL-2Rβ and IL-2Rγ) and the binding of an antigen-binding molecule to a specific antigen (e.g., CD8 or PD-1).

[0064] "Mutant IL-2 polypeptide" refers to an IL-2 polypeptide that has reduced affinity for its receptor, such a reduction in affinity resulting in reduced biological activity of the mutant. Reduced affinity and therefore reduced activity can be achieved by introducing a small number of amino acid mutations or substitutions. Mutant IL-2 polypeptides may also have other modifications to the peptide backbone to generate a final construct with desired properties, such as reduced affinity for IL-2Rβγ, including, but not limited to, amino acid deletion, rearrangement, cyclization, disulfide bonds or post-translational modifications (e.g., glycosylation or carbohydrate alterations) of the polypeptide, chemical or enzymatic modifications to the polypeptide (e.g., attachment of PEG to the polypeptide backbone), addition of peptide tags or labels, or fusion to proteins or protein domains. Desired activity can also include improved biophysical properties compared to wild-type IL-2 polypeptide. Multiple modifications may be combined to achieve desired activity modifications, such as reduced affinity or improved biophysical properties. As a non-limiting example, an amino acid sequence for consensus N-linked glycosylation can be incorporated into the polypeptide to allow for glycosylation. Another non-limiting example is that a lysine can be incorporated onto the polypeptide to allow for PEGylation. Preferably, a mutation(s) is introduced into the polypeptide to modify its activity.

[0065] "Targeting moiety" and "antigen-binding molecule" as used herein in their broadest sense refer to a molecule that specifically binds to an antigenic determinant. A targeting moiety or antigen-binding molecule can be a protein, carbohydrate, lipid or other compound. A targeting moiety or antigen-binding molecule can be an antibody, an antibody fragment (Chames et al, 2009; Chan & Carter, 2010; Leavy, 2010; Holliger & Hudson, 2005), a scaffold antigen-binding protein (Gebauer and Skerra, 2009; Stumpp et al, 2008), a single domain antibody (sdAb), a minibody (minAb), or a fusion protein (FcAb). ibody (Tramontano et al., 1994), variable domain of heavy chain antibody (nanobody ( nanobody), VHH), variable domain of novel antigen receptor (VNAR), carbohydrate-binding domain (CBD) (Blake et al., 2006), collagen-binding domain (Knight et al., 2000), lectin-binding protein (tetranectin), collagen-binding protein Protein, Adnectin / fibronectin (Lipovsek, 2011), serum transferrin ( trans-bodies), Evibodies, Protein A derived molecules such as the Z domain of Protein A (Affibodies) (Nygren et al, 2008), the A domain (Avimers / maxibodies) (Maxibody), alphabody (WO2010066740), avimer / maxibody, designed ankyrin repeat domains (DARPins) (Stumpp et al, 2008), anticalins (Skerra et al, 2008), human gamma crystallin or ubiquitin (Affilin molecules), Kunitz-type domains of human protease inhibitors, knottins (Kolmar et al, 2008), linear or constrained peptides with or without fusions to extend half-life, e.g. For example, (Fc fusions-peptibodies) (Rentero Rebollo & Heinis, 2013; EP1144454B2; Shimamoto et al., 2012; US7205275B2), constrained bicyclic peptides (US2018 / 0200378A1), aptamers, engineered CH2 domains (nanobodies; Dimitrov, 2009) and engineered CH3 domains. These include, but are not limited to, the in “Fcab” domain (Wozniak-Knopp et al, 2010).

[0066] The terms "antibody" and "immunoglobulin" are used interchangeably and are used herein in the broadest sense to encompass a variety of antibody structures, including, but not limited to, monoclonal antibodies (e.g., full-length or intact monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, and single domain antibodies (as described in more detail herein), so long as they exhibit the desired antigen-binding activity.

[0067] An antibody (immunoglobulin) refers to a protein having a structure substantially similar to that of a natural antibody. A "natural antibody" refers to a naturally occurring immunoglobulin molecule having a variety of structures. For example, a natural immunoglobulin of the IgG class is a heterotetrameric glycoprotein of about 150,000 daltons, composed of two light chains and two heavy chains that are disulfide-bonded. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also called the variable heavy domain or the heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), also called the heavy chain constant region. Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also called the variable light domain or the light chain variable domain, followed by a constant light (CL) domain, also called the light chain constant region. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are described, for example, in Abbas et al., 2000, Cellular and Mol, and Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007). Antibodies (immunoglobulins) are assigned to different classes depending on the amino acid sequence of the heavy chain constant domain. There are five major classes of antibodies: α (IgA), δ (IgD), ε (IgE), γ (IgG) or μ (IgM), some of which can be further divided into subtypes, e.g., γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1) and α2 (IgA2). The light chain of an immunoglobulin can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain. Immunoglobulins essentially consist of two Fab molecules and an Fc domain, linked via an immunoglobulin hinge region.

[0068] "Fc" or "Fc region" or "Fc domain" as used herein refers to the C-terminal region of an antibody heavy chain that contains at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and optionally all or a portion of the flexible hinge N-terminal to these domains. For IgA and IgM, Fc can include the J chain. The IgG Fc region is the C-terminal region of an IgG The Fc region includes the CH2 and IgG CH3 domains, in some cases including the hinge. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is in accordance with Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., The EU numbering system, also referred to as the EU index, as described in 1991. The "hinge" region usually extends from about amino acid residue 216 to about amino acid residue 230. The hinge region may be a native or variant hinge domain herein. The "CH2 domain" of a human IgG Fc region usually extends from about amino acid residue 231 to about amino acid residue 340. The CH2 domain may be a native or variant CH2 domain herein. The "CH3 domain" comprises the stretch of residues C-terminal to the CH2 domain in the Fc region, from about amino acid residue 341 to about amino acid residue 447 of IgG. The CH3 region, as used herein, may be a native sequence CH3 domain or a variant CH3 domain (e.g., a CH3 domain in which a "protrusion" ("knob") is introduced in one of its chains and a corresponding "cavity" ("hole") is introduced in the other chain; see U.S. Pat. No. 5,821,333, expressly incorporated herein by reference). Thus, the definition of "Fc domain" includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or fragments thereof. An "Fc fragment" in this context may contain fewer amino acids from either or both the N-terminus and C-terminus, but still retain the ability to form a dimer with another Fc domain or Fc fragment, as may be detected using standard methods, generally based on size (e.g., non-denaturing chromatography, size exclusion chromatography, etc.). Human IgG Fc domains are particularly useful in the present disclosure and may be Fc domains from human IgG1, IgG2, or IgG4.

[0069] A "variant Fc domain" or "Fc variant" or "variant Fc" has amino acid modifications (e.g., substitutions, additions and deletions) compared to a parent Fc domain. The term also includes naturally occurring allelic variants of the Fc region of an immunoglobulin. Generally, a variant Fc domain has at least about 80, 85, 90, 95, 97, 98 or 99 percent identity (using the identity algorithm discussed below, with one embodiment utilizing the BLAST algorithm as known in the art, using default parameters) to a corresponding parent human IgG Fc domain. Alternatively, a variant Fc domain may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acid modifications compared to the parent Fc domain. For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the Fc region of an immunoglobulin without significant loss of biological function. In addition, as discussed herein, the variant Fc domains herein still retain the ability to form dimers with another Fc domain, as measured using known techniques described herein, such as non-denaturing gel electrophoresis.

[0070] By "Fc gamma receptor", "FcγR" or "Fc gamma R" as used herein is meant any member of a family of proteins that bind to the IgG antibody Fc region and are encoded by the FcγR gene. In humans, this family includes, but is not limited to, FcγRI (CD64), which includes isoforms FcγRIa, FcγRIb, and FcγRIc; FcγRII (CD32), which includes isoforms FcγRIIa (including allotypes H131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), and FcγRIIc; and FcγRIII (CD16), which includes isoforms FcγRIIIa (including allotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1 and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, incorporated by reference in its entirety); and any undiscovered human FcγR or FcγR isoform or allotype. FcγR can be from any organism, including, but not limited to, human, mouse, rat, rabbit and monkey. Mouse FcγR includes, but is not limited to, FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIII-2 (CD16-2), and any undiscovered mouse FcγR or FcγR isoform or allotype.

[0071] "Effector function" as used herein refers to a biochemical event resulting from the interaction of an antibody Fc region with an Fc receptor or ligand, which may vary depending on the antibody isotype. Effector functions include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), complement-dependent cytotoxicity (CDC), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation. "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibody on a target cell and subsequently cause the lysis of the target cell. ADCC correlates with binding to FcγRIIIa, and increased binding to FcγRIIIa leads to increased ADCC activity. To assess the ADCC activity of a molecule of interest, an in vitro ADCC assay can be performed, such as that described in U.S. Patent No. 5,500,362 or 5,821,337. "ADCP" or antibody-dependent cell-mediated phagocytosis, as used herein, refers to a cell-mediated reaction in which non-specific cytotoxic cells expressing FcγR recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0072] "Fc null" and "Fc null variant" are used interchangeably and are used herein to describe modified Fc with reduced or eliminated effector function. Such Fc null or Fc null variant has reduced or eliminated FcγR and / or complement receptor. Preferably, such Fc null or Fc null variant has eliminated effector function. Exemplary methods for this modification include, but are not limited to, chemical modification, amino acid residue substitution, insertion and deletion. Exemplary amino acid positions on the Fc molecule where one or more modifications were introduced at positions (numbering according to the EU numbering scheme) to reduce effector function of the resulting variants: i) IgG1: C220, C226, C229, E233, L234, L235, G237, P238, S239 D265, S267, N297, L328, P331, K322, A327 and P329, ii) IgG2: V234, G237, D265, H268, N297, V309, A330, A331, K322, and iii) IgG4: L235, G237, D265 and E318.Exemplary Fc molecules with reduced effector function include those with one or more of the following substitutions: i) IgG1: N297A, N297Q, D265A / N297A, D265A / N297Q, C220S / C226S / C229S / P238S, S267E / L328F, C226S / C229S / E233P / L234V / L235A, L235A / L235B, L235C / L235C ... 34F / L235E / P331S, L234A / L235A, L234A / L235A / G237A, L234A / L235A / G237A / K322A, L234A / L235A / G2 37A / A330S / A331S, L234A / L235A / P329G, E233P / L234V / L235A / G236del / S239K, E233P / L234V / L235A / G 236del / S267K, E233P / L234V / L235A / G236del / S239K / A327G, E233P / L234V / L235A / G236del / S267K / A 327G and E233P / L234V / L235A / G236del, L234A / L235A / G237 deletion; ii) IgG2:A330S / A331S, V234A / G237A, V 234A / G237A / D265A, D265A / A330S / A331S, V234A / G237A / D265A / A330S / A331S, and H268Q / V309L / A330S / A331S; iii) IgG4: L235A / G237A / E318A, D265A, L235A / G237A / D265A and L235A / G237A / D265A / E318A.

[0073] "Epitope" as used herein refers to a determinant capable of specific binding to a variable region of an antibody molecule known as a paratope. An epitope is a grouping of molecules such as amino acids or sugar side chains, which usually have specific structural as well as charge characteristics. A single antigen may have more than one epitope. An epitope may include amino acid residues that are directly involved in binding and other amino acid residues that are not directly involved in binding, e.g., amino acid residues that are effectively blocked by the antigen-binding peptide (in other words, they are within the footprint of the antigen-binding peptide). An epitope may be either a conformational epitope or a linear epitope. An epitope typically includes at least 3, more usually at least 5 or 8-10 amino acids. Antibodies that recognize the same epitope can be verified in a simple immunoassay, e.g., "binning", which reveals the ability of one antibody to block the binding of another antibody to a target antigen.

[0074] "Linker" as used herein refers to a molecule that connects two polypeptide chains. The linker may be a polypeptide linker or a synthetic chemical linker (see, for example, those disclosed in Protein Engineering, 9(3), 299-305, 1996). The length and sequence of the polypeptide linker are not particularly limited and may be selected according to purpose by those skilled in the art. The polypeptide linker comprises one or more amino acids. Preferably, the polypeptide linker is a peptide having a length of at least 5 amino acids, preferably 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is G, S, GS, SG, SGG, GGS and GSG (G=glycine and S=serine). In another embodiment, the peptide linker is (GGGS)xGn (SEQ ID NO:5) or (GGGGS)xGn (SEQ ID NO:6) or (GGGGGS)xGn (SEQ ID NO:7) (x=1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and n=0, 1, 2 or 3). Preferably, the linker is (GGGGS)xGn (x=2, 3 or 4, and n=0) (SEQ ID NO:8), more preferably, the linker is (GGGGS)xGn (x=3, and n=0) (SEQ ID NO:9). Synthetic chemical linkers include cross-linkers routinely used to cross-link peptides, such as N-hydroxysuccinimide (NHS), disuccinimidyl suberate (DSS), bis(succinimidyl) suberate (BS3), dithiobis(succinimidyl propionate) (DSP), dithiobis(succinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), ethylene glycol bis(sulfosuccinimidyl succinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).

[0075] The term "polynucleotide" refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virus-derived RNA, or plasmid DNA (pDNA), encoding a polypeptide of the present disclosure. A polynucleotide may contain conventional phosphodiester bonds or may contain non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNAs)). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. In some aspects, one or more vectors (particularly expression vectors) containing such nucleic acids are provided. In one aspect, a method for making a polypeptide of the present disclosure is provided, comprising culturing a host cell containing a nucleic acid encoding the polypeptide under conditions suitable for expression of the polypeptide, and recovering the polypeptide from the host cell. "Recombinant" refers to a protein produced using recombinant nucleic acid techniques in an exogenous host cell. Recombinantly produced proteins expressed in a host cell are considered isolated in the present disclosure, and natural or recombinant proteins that have been separated, fractionated, or partially or substantially purified by any suitable technique are also considered isolated.

[0076] "Isolated" when used to describe various polypeptides disclosed herein means a polypeptide that has been identified and separated and / or recovered from a cell or cell culture in which the polypeptide is expressed. Typically, an isolated polypeptide will be purified by at least one purification step. There is no requirement for the level of purification, and "purification" or "purified" refers to an increase in the concentration of a target protein relative to the concentration of contaminants in a composition compared to the starting material. "Isolated protein" as used herein refers to a target protein that is substantially free of other proteins with different binding specificities.

[0077] The term "cancer" refers to a physiological condition in mammals that is typically characterized by uncontrolled and abnormal cell growth that may invade or spread to other parts of the body. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specific examples of such cancers include lung cancer, small cell lung cancer, non-small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, head and neck cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, skin and intraocular melanoma, thyroid cancer, uterine cancer, gastrointestinal cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, gastric cancer, and gastric cancer. cancer, colon cancer, breast cancer, endometrial cancer, uterine cancer, fallopian tube cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer, mesothelioma, bladder cancer, liver cancer, hepatoma, hepatocellular carcinoma, cervical cancer, salivary gland cancer, bile duct cancer, central nervous system (CNS) neoplasms, spinal axis tumor, brain stem glioma, glioblastoma multiforme, astrocytoma, schwanoma, ependymona, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and Ewing's sarcoma, including refractory versions of any of the above cancers, or a combination of one or more of the above cancers.

[0078] Mutant IL-2 Polypeptides The present disclosure provides, inter alia, mutant IL-2 polypeptides that exhibit less than 50% binding affinity to IL-2Rα (e.g., comprising an amino acid sequence of SEQ ID NO:2 or as shown in FIG. 1B). In some embodiments, the mutant IL-2 polypeptides also exhibit less than 50% binding affinity to IL-2Rβ (e.g., comprising an amino acid sequence of SEQ ID NO:3 or as shown in FIG. 1C). In some embodiments, the mutant IL-2 polypeptides exhibit less than 50% binding affinity to IL-2Rα and less than 50% binding affinity to IL-2Rβ (e.g., comprising an amino acid sequence of SEQ ID NO:3 or as shown in FIG. 1C) compared to wild-type IL-2 polypeptides (e.g., comprising an amino acid sequence of SEQ ID NO:1 or as shown in FIG. 1A). In some embodiments, the mutant IL-2 polypeptides exhibit less than 50% binding affinity to IL-2Rα and less than 50% binding affinity to IL-2Rγ (e.g., comprising an amino acid sequence of SEQ ID NO:4 or as shown in FIG. 1D) compared to wild-type IL-2 polypeptides (e.g., comprising an amino acid sequence of SEQ ID NO:1 or as shown in FIG. 1A). In some embodiments, the mutant IL-2 polypeptides exhibit less than 50% binding affinity to IL-2Rα, less than 50% binding affinity to IL-2Rβ, and less than 50% binding affinity to IL-2Rγ compared to wild-type IL-2 polypeptides. The difference in binding affinity of wild-type and mutant polypeptides of the present disclosure to IL-2Rα and IL-2Rβ can be measured, for example, by standard surface plasmon resonance (SPR) assays that measure the affinity of protein-protein interactions, which are well known to those of skill in the art. The difference in binding affinity of wild-type and mutant polypeptides of the present disclosure to IL-2Rγ cannot be reliably measured by SPR assays because the affinity of wild-type IL-2 polypeptides to IL-2Rγ is very low. Instead, their reduced affinity to IL-2Rγ can be estimated by performing in vitro assays that measure pSTAT5 and compare the activity of IL-2 polypeptides with and without substitutions that reduce affinity to IL-2Rγ on IL-2R-expressing cells.

[0079] The mutant IL-2 polypeptides of the disclosure have one or more, two or more, or three or more affinity-reducing amino acid substitutions relative to a wild-type mature IL-2 polypeptide having an amino acid sequence as depicted in Figure 1A (SEQ ID NO:1), where the one or more, two or more, or three or more substituted residues are selected from the following group: Q11, H16, L18, L19, D20, D84, S87, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, D84, S87, N88, V91, I92, T123, Q126, S127, I129, and S130. The locations of possible amino acid substitutions within the sequence of a wild-type mature IL-2 polypeptide are depicted, for example, in Figure 2. A reduced affinity for IL-2Rα can be achieved by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: R38, F42, K43, Y45, E62, P65, E68, V69 and L72. A reduced affinity for IL-2Rβ can be achieved by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: E15, H16, L19, D20, D84, S87, N88, V91 and I92. A reduced affinity for IL-2Rγ can be achieved by substituting one or more of the following residues in the sequence of the wild-type mature IL-2 polypeptide: Q11, L18, Q22, T123, Q126, S127, I129 and S130.

[0080] In some embodiments, a mutant IL-2 polypeptide comprises an F42A or F42K amino acid substitution relative to the wild-type mature IL-2 amino acid sequence, e.g., as depicted in Figure 1A (SEQ ID NO: 1). In some embodiments, a mutant IL-2 polypeptide comprises an F42A or F42K amino acid substitution and an R38A, R38D, R38E, E62Q, E68A, E68Q, E68K or E68R amino acid substitution relative to the wild-type mature IL-2 amino acid sequence, e.g., as depicted in Figure 1A (SEQ ID NO: 1). For example, in some embodiments, a mutant IL-2 polypeptide comprises an F42A; R38A and F42A; R38D and F42A; R38E and F42A; F42A and E62Q; F42A and E68A; F42A and E68Q; F42A and E68K; F42A and E68R; or an R38A and F42K amino acid substitution relative to the wild-type mature IL-2 amino acid sequence, e.g., as depicted in FIG. 1A (SEQ ID NO:1). In some embodiments, a mutant IL-2 polypeptide comprises an R38E and F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, a mutant IL-2 polypeptide comprises an R38D and F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, a mutant IL-2 polypeptide comprises an F42A and E62Q amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises an R38A and an F42K amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises an R38D and an F42A amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises an R38A and an F42K amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises an F42A and an E62Q amino acid substitution relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises an H16E, H16D, D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88S, N88T,Includes N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K or S127Q amino acid substitutions. In some embodiments, the mutant IL-2 polypeptides include F42A; R38A and F42A; R38D and F42A; R38E and F42A; F42A and E62Q; F42A and E68A; F42A and E68Q; F42A and E68K; F42A and E68R; or R38A and F42K amino acid substitutions relative to the wild-type mature IL-2 amino acid sequence depicted in FIG. 1A (SEQ ID NO:1), and including H16E, H16D, D20N, M23A, M23R, M23K, D84L, D84N, D84V, D84H, D84Y, D84R, D84K, S87K, S87A, N88A, N88S, N88T, N88R, N88I, V91A, V91T, V91E, I92A, E95S, E95A, E95R, T123A, T123E, T123K, T123Q, Q126A, Q126S, Q126T, Q126E, S127A, S127E, S127K or S127Q amino acid substitutions. For example, in some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and H16E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and H16D amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and N88S amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and N88A amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and V91E amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments, the mutant IL-2 polypeptide comprises R38E, F42A and Q126S amino acid substitutions relative to the wild-type IL-2 amino acid sequence. In some embodiments,The mutant IL-2 polypeptides comprise the following sets of amino acid substitutions (relative to the sequence of SEQ ID NO:1): R38E and F42A; R38D and F42A; F42A and E62Q; R38A and F42K; R38E, F42A and N88S; R38E, F42A and N88A; R38E, F42A and V91E; R38E, F42A and D84H; H16D, R38E and F42A; H16E, R38E and F42A; R38E, F42A and Q126S; R38D, F42A and N88S; R38D, F42A and N88A; R38D, F42A and V91E;R38D, F42A and D84H;H16D, R38D and F42A;H16E, R38D and F42A;R38D, F42A and Q126S;R38A, F42K and N88S;R38A, F42K and N88A;R38A, F42K and V91E;R38A, F42K and D84H;H16D, R38A and F42K;H16E, R38A and F42K;R38A, F42K and Q126S;F42A, E62Q and N88S;F42A, E62Q and N88A;F42A, E62Q and V91 E;F42A,E62Q and D84H;H16D,F42A and E62Q;H16E,F42A and E62Q;F42A,E62Q and Q126S;R38E,F42A and C125A;R38D,F42A and C125A;F42A,E62Q and C125A;R38A,F42K and C125A;R38E,F42A,N88S and C125A;R38E,F42A,N88A and C125A;R38E,F42A,V91E and C125A;R38E,F42A,D84H and C125A;H16D,R38E,F42A and and C125A;H16E, R38E, F42A and C125A;R38E, F42A, C125A and Q126S;R38D, F42A, N88S and C125A;R38D, F42A, N88A and C125A;R38D, F42A, V91E and C125A;R38D, F42A, D84H and C125A;H16D, R38D, F42A and C125A;H16E, R38D, F42A and C125A;R38D, F42A, C125A and Q126S;R38A, F42K, N88S and C125A;R38A, F42K,N88A and C125A;R38A, F42K, V91E and C125A;R38A, F42K, D84H and C125A;H16D, R38A, F42K and C125A;H16E, R38A, F42K and C125A;R38A, F42K, C125A and Q126S;F42A, E62Q, N88S and C125A;F42A, E62Q, N88A and C125A;F42A, E62Q, V91E and C125A;F42A, E62Q and D84H and C125A;H16 and F42A, C125A and Q126S; F42A, N88S and C125A; F42A, N88A and C125A; F42A, V91E and C125A; F42A, D84H and C125A; H16D, F42A and C125A; H16E, F42A and C125A; and F42A, C125A and Q126S. In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 18). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 19). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 20).The mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 21). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 22). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 23). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 24). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 25).APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 26). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEEL, KPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 27). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 28). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 29). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 30). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 31). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 32).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 33). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 34). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 35). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 36). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 37).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 38). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 39). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 40). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 41). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 42).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 43). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 44). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 45). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 46). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (SEQ ID NO: 47).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 48). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCSSIISTLT (SEQ ID NO: 49). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 50). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 51). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:52).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 53). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 54). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 55). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKAT. In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 56). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 57). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 58). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 59). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTEMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 60). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 61).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 62). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 63). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 64). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 65). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 66).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTDMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 67). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 68). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 69). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 70). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:71).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 72). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 73). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTAMLTKKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 74). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 75). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO:76).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 77). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRHLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 78). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 79). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 80). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEQLKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 81).In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISSINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 82). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISAINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 83). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINEIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 84). In some embodiments, the mutant IL-2 polypeptide comprises APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM. In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEDLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 86). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEELLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFAQSIISTLT (SEQ ID NO: 87). In some embodiments, the mutant IL-2 polypeptide comprises the amino acid sequence of APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTAKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFASSIISTLT (SEQ ID NO: 88).

[0081] In some embodiments, the mutant IL-2 polypeptides of the present disclosure also contain other modifications, including but not limited to mutations and deletions, that provide additional benefits, such as improved biophysical properties. Improved biophysical properties include but are not limited to improved thermostability, aggregation properties, acid reversibility, viscosity, and production in mammalian or bacterial or yeast cells. For example, residue C125 can be replaced with a neutral amino acid, such as serine, alanine, threonine, or valine, and the N-terminal A1 residue can be deleted, both of which are described in U.S. Pat. No. 4,518,584. The mutant IL-2 polypeptides may also include a mutation of residue M104, such as M104A, as described in U.S. Pat. No. 5,206,344. Thus, in certain embodiments, the mutant IL-2 polypeptides of the present disclosure include the amino acid substitution C125A. In other embodiments, one, two, or three N-terminal residues are deleted.

[0082] Fusion proteins The present disclosure provides a fusion protein comprising a mutant IL-2 polypeptide of the disclosure and an antigen binding molecule that binds to one of the following antigens: CD8α, CD8β, and PD1, wherein the fusion protein preferentially activates immune cells that express an antigen against the antigen binding molecule of the fusion over immune cells that do not express the antigen.

[0083] The preferential activity of targeted IL-2 fusion proteins comprising mutant IL-2 polypeptides against antigen-expressing cells is demonstrated in assays involving antigen-expressing and non-antigen-expressing cells that also express IL-2Rβγ or IL-2Rαβγ. One such assay is an in vitro assay that measures STAT5 (pSTAT5) phosphorylation and / or expression of the proliferation marker Ki-67 in human immune cells, such as human peripheral blood and / or tumor-infiltrating immune cells, upon exposure to IL-2 polypeptides. In one format of the assay, the activity of targeted IL-2 fusion proteins is measured against antigen-expressing and non-antigen-expressing cells to demonstrate selectivity for antigen-expressing cells. In another format of the assay, the activity of targeted IL-2 fusion proteins comprising mutant IL-2 polypeptides against antigen-expressing cells is compared to the activity of non-targeted IL-2 fusion proteins comprising the same mutant IL-2 polypeptides and a control antibody that does not recognize any antigen on antigen-expressing cells to demonstrate the magnitude of rescue in signaling of the mutant IL-2 polypeptides when fused to an antigen-binding molecule.

[0084] In some embodiments, a fusion protein of the present disclosure containing a CD8α antigen binding molecule activates CD8α+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold over CD8α-IL-2Rβ+ cells. In some embodiments, the fusion protein activates CD8α+IL-2Rβ+ cells more than 50-fold, 100-fold, or 200-fold compared to a fusion molecule comprising the IL-2 variant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation by an IL-2 fusion protein is determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in the cells after treatment with the IL-2 fusion protein.

[0085] In some embodiments, a fusion protein of the present disclosure containing a CD8β antigen binding molecule activates CD8β+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold over CD8β-IL-2Rβ+ cells. In some embodiments, the fusion protein activates CD8β+IL-2Rβ+ cells by more than 50-fold, 100-fold, or 200-fold compared to a fusion molecule comprising the IL-2 variant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation by an IL-2 fusion protein is determined by measuring the expression of pSTAT5 or the cell proliferation marker Ki67 in the cells after treatment with the IL-2 fusion protein.

[0086] In some embodiments, a fusion protein of the disclosure containing a PD1 antigen binding molecule activates PD1+IL-2Rβ+ cells at least 10-fold, at least 50-fold, or at least 100-fold over PD1-IL-2Rβ+ cells. In some embodiments, the fusion protein activates PD1+IL-2Rβ+ cells by more than 50-fold, 100-fold, or 200-fold compared to a fusion protein comprising the IL-2 variant polypeptide and a control antibody that does not bind to any antigen expressed on the cells. The cell activation by an IL-2 fusion protein is determined by measuring expression of pSTAT5 or the cell proliferation marker Ki67 in the cells following treatment with the IL-2 fusion protein.

[0087] In some embodiments, the fusion proteins of the present disclosure exhibit one or more of the following: bind to human CD8 and do not block the interaction of CD8 with MHC class I; and activate CD8+ T cells with at least 10-fold, 25-fold, 50-fold, 100-fold, 250-fold, 500-fold, or 1000-fold greater potency, for example, compared to the activation of NK cells. In some embodiments, whether an anti-CD8 antibody or fusion protein of the present disclosure blocks the interaction of CD8 with MHC class I can be assayed, for example, by assaying the activation status (e.g., by antigen stimulation) of CD8+ T cells in the presence or absence of the anti-CD8 antibody or fusion protein. For exemplary assays and conditions, see, for example, Example 3. In some embodiments, the activation of CD8+ T cells and / or NK cells can be measured, for example, by assaying one or more markers (e.g., the proportion of treated cells expressing one or more markers) of proliferation (e.g., Ki67), IL-2Rβ / γ downstream signaling, and / or STAT5 downstream signaling. See, eg, Example 5 for exemplary assays and conditions.

[0088] By extension of these findings, the fusion proteins of the present disclosure can contain a polypeptide that binds to IL-2Rαβγ, where the binding affinity to IL2Rα has been reduced by methods other than introducing a small number of mutations into the sequence of the wild-type IL-2 polypeptide. Thus, the fusion proteins of the present invention can include IL-2 polypeptides fused to IL-2Rα, as described in Lopes et al, J Immunother Cancer. 2020; 8(1): e000673. or by using synthetic polypeptide mimetics that are computationally designed to bind IL-2Rβγ but not IL-2Rα, such as those described in Silva et al, Nature. 2019 Jan;565(7738):186-191; or antigen-binding domain polypeptides that are agonists of IL-2Rβγ. Such polypeptides can be fused to CD8 antibodies to construct fusions that result in a 10-fold or greater selective potency of those fusions for CD8+ T cells compared to NK cells.

[0089] Fusion Protein Formats The fusion proteins have different formats as depicted in Figure 4. In some embodiments, the fusion protein comprises two moieties as depicted in Figure 4A: i) the first moiety is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer (wherein VH is the variable heavy chain and CH2-CH3 is the Fc domain), an antibody light chain VL-CL (wherein VL is the variable light chain and CL is the constant light chain) and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second moiety is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; both the first moiety and the second moiety bind to an epitope on one antigen selected from the following group: human CD8α, human CD8β and human PD1.

[0090] In some embodiments, the fusion protein comprises two portions as depicted in FIG. 4B: i) the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer (wherein CH2-CH3 is an Fc domain) and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL, wherein the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1.

[0091] In some embodiments, the fusion protein comprises two portions as depicted in FIG. 4C: i) the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer (wherein CH2-CH3 is an Fc domain) and a mutant IL-2 polypeptide, wherein the C-terminus of the mutant IL-2 polypeptide is fused to the N-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; and the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1.

[0092] In some embodiments, the first and second Fc domains of the fusion protein contain the following Fc mutations according to EU numbering to reduce effector function: L234A, L235A, G237A and K322A. In some embodiments, the first and second Fc domains of the fusion protein contain the following Fc mutations according to EU numbering to reduce effector function: L234A, L235A, G237A and K322A. In some embodiments, the first and second Fc domains of the fusion protein contain the following amino acid substitutions to promote heterodimer formation: Y349C / T366W (knob) and S354C, T366S, L368A and Y407V (hole).

[0093] In some embodiments, the recombinant bispecific antibodies and / or fusion proteins disclosed herein can be very broadly classified into two categories: i) formats resulting from the combination of only variable regions, and ii) formats combining variable regions and Fc domains. Representatives of the first category are tandem scFvs (taFvs), diabodies (Dbs), DARTs, single chain diabodies (scDbs), Fab-Fcs, tandem Fabs, dual variable region Fabs, and tandem dAbs / VHHs. The two variable regions may be linked together by covalent bonds or by non-covalent interactions.

[0094] In some embodiments, bispecific antibodies / fusion proteins are generated in the natural immunoglobulin structure, containing two pairs of heavy and light chain combinations, each pair having a distinct binding specificity. The homodimerization of the two heavy chains in IgG is mediated by CH3 interactions. To promote the formation of heterodimers, genetic modifications are introduced into the two respective CH3 regions. The heterodimerization mutations there often involve steric repulsion, charge-induced interactions, or interchain disulfide bond formation. Exemplary Fc modifications to promote heterodimerization include, but are not limited to, the following: [Table A-1] [Table A-2]

[0095] In some embodiments, bispecific antibodies can be produced by post-production assembly from half antibodies, which solves the problem of mispairing of heavy and light chains. These antibodies often contain modifications that favor heterodimerization of the half antibodies. Exemplary systems include, but are not limited to, knob-into-hole, IgG1 (EEE-RRR), IgG2 (EEE-RRRR) (Strop et al. J Mol Biol (2012)) and DuoBody (F405L-K409R), which are listed in Table A. In such cases, the half antibodies are individually produced and purified in separate cell lines. The purified antibodies are then subjected to mild reduction to obtain half antibodies, which are then assembled into bispecific antibodies. The heterodimeric bispecific antibodies are then purified from the mixture using conventional purification methods.

[0096] In some embodiments, strategies using bispecific antibody generation that do not rely on selective pairing of chains can also be used. These strategies typically involve introducing genetic modifications to the antibody such that the heterodimer will have distinctly different biochemical or biophysical properties than the homodimer, and thus the heterodimer after assembly or expression can be selectively purified from the homodimer. One example is introducing H435R / Y436F in the IgG1 CH3 domain to abolish Fc binding to Protein A resin, and then co-expressing the H435R / Y436F variant with wild-type Fc. The resulting homodimeric antibody containing two copies of H435R / Y436F will not be able to bind to a Protein A column, while a heterodimeric antibody containing one copy of the H435R / Y436F mutation will have a reduced affinity for Protein A compared to the strong interaction from the homodimeric wild-type antibody (Tustian et al Mabs 2016). Another example is the introduction of H435R / Y436F in the IgG1 CH3 domain to abolish Fc binding to Protein A resin, and then co-expressing the H435R / Y436F variant with wild-type Fc. The resulting homodimeric antibody containing two copies of H435R / Y436F will not be able to bind to a Protein A column, while a heterodimeric antibody containing one copy of the H435R / Y436F mutation will have a reduced affinity for Protein A compared to the strong interaction from the homodimeric wild-type antibody (Tustian et al Mabs 2016). These include α / lambda antibodies (Fischer et al., Nature Communication 2015), and the introduction of different charges (E357Q, S267K or N208D / Q295E / N384D / Q418E / N421D) into each chain (US2018 / 0142040A1; Strop et al. J Mol Biol (2012)).

[0097] In some embodiments, bispecific antibodies can be generated by fusing additional binding sites to either the heavy or light chain of the immunoglobulin. Examples of additional binding sites include, but are not limited to, variable regions, scFv, Fab, VHH, and peptides.

[0098] In some embodiments, heterodimer mutations and / or mutations to modify Fc gamma receptor binding resulted in a reduction in Fc stability. Thus, additional mutations were added to the Fc region to increase its stability. For example, one or more disulfide bond pairs are introduced into the Fc region, such as A287C and L306C, V259C and L306C, R292C and V302C, and V323C and I332C. Another example is the introduction of S228P into an IgG4-based bispecific antibody to stabilize hinge disulfides. Additional examples include the introduction of K338I, A339K, and K340S mutations to enhance Fc stability and aggregation resistance (Gao et al, 2019 Mol Pharm. 2019;16:3647). can be.

[0099] antigen binding molecule In some embodiments, the fusion protein binds to human CD8, and the binding of the fusion protein to CD8 does not block the interaction of CD8 with MHC class I. In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on CD8α, and the binding of the antigen binding molecule to CD8α does not block the interaction of CD8αα or CD8αβ with MHC class I molecules on target cells or antigen-presenting cells. In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on CD8β, and the binding of the antigen binding molecule to CD8β does not block the interaction of CD8αβ with MHC class I molecules on target cells or antigen-presenting cells. In some embodiments, whether an anti-CD8 antibody or fusion protein of the present disclosure blocks the interaction of CD8 with MHC class I can be assayed, for example, by assaying the activation status (e.g., by antigen stimulation) of CD8+ T cells in the presence or absence of the anti-CD8 antibody or fusion protein. For exemplary assays and conditions, see, for example, Example 3.

[0100] In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVQLVESGGGLVQPGRSLKLSCAASGFTFSNYYMAWVRQAPTKGLEWVAYINTGGGTTYYRDSVKGRFTISRDDAKSTLYLQMDSLRSEDTATYYCTTAIGYYFDYWGQGVMVTVSS (SEQ ID NO: 10) and a VL domain comprising the sequence of DIQLTQSPASLSASLGETVSIECLASEDIYSYLAWYQQKPGKSPQVLIYAANRLQDGVPSRFSGSGSGTQYSLKISGMQPEDEGDYFCLQGSKFPYTFGAGTKLELK (SEQ ID NO: 11). In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVKLQESGPSLVQPSQTLSLTCSVSGFSLISDSVHWVRQPPGKGLEWMGGIWADGSTDYNSALKSRLSISRDTSKSQGFLKMNSLQTDDTAIYFCTSNRESYYFDYWGQGTMVTVSS (SEQ ID NO: 12) and a VL domain comprising the sequence of DIQMTQSPASLSASLGDKVTITCQASQNIDKYIAWYQQKPGKAPRQLIHYTSTLVSGTPSRFSGSGSGRDYSFSISSVESEDIASYYCLQYDTLYTFGAGTKLELK (SEQ ID NO: 13). In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVKLQESGPSLVQPSQTLSLTCSVSGFSLISDSVHWVRQPPGKGLEWMGGIWADGSTDYNSALKSRLSISRDTSKSQGFLKMNSLQTDDTAIYFCTSARESYYFDYWGQGTMVTVSS (SEQ ID NO: 14) and a VL domain comprising the sequence of DIQMTQSPASLSASLGDKVTITCQASQNIDKYIAWYQQKPGKAPRQLIHYTSTLVSGTPSRFSGSGSGRDYSFSISSVESEDIASYYCLQYATLYTFGAGTKLELK (SEQ ID NO: 15).In some embodiments, an anti-CD8 antibody or fusion protein of the disclosure comprises a VH domain comprising the sequence of EVQLVESGGALVQPGRSLKLSCAASGLTFSDCYMAWVRQTPTKGLEWVSYISSDGGSTYYGDSVKGRFTISRDNAKSTLYLQMNSLRSEDMATYYCACATDLSSYWSFDFWGPGTMVTVSS (SEQ ID NO: 16) and a VL domain comprising the sequence of DIQMTQSPSSLPVSLGERVTISCRASQGISNNLNWYQQKPDGTIKPLIYHTSNLQSGVPSRFSGSGSGTDYSLTISSLEPEDFAMYYCQQDATFPLTFGSGTKLEIK (SEQ ID NO: 17).

[0101] In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on PD1, and the binding of the antigen binding molecule to PD1 does not block the interaction of PD1 with PD-L1 expressed on target cells or other immune cells. Such fusion proteins are particularly useful because they can be administered as therapeutic agents in combination with anti-PD1 therapeutic antibodies, including but not limited to nivolumab, pembrolizumab, and cemiplimab.

[0102] In some embodiments, the antigen binding molecule of the present disclosure binds to an epitope on PD1, and the binding of the antigen binding molecule to PD1 blocks the interaction of PD1 with PD-L1 on target cells or other immune cells. Such fusion proteins are particularly useful because they can be administered as therapeutic agents in combination with anti-PDL1 therapeutic antibodies, including but not limited to atezolizumab, avelumab, and durvalumab.

[0103] Certain aspects of the present disclosure relate to methods for treating cancer or chronic infections. In some embodiments, the methods include administering to a patient an effective amount of a fusion protein or a pharmaceutical composition comprising the fusion protein and a pharma- ceutically acceptable carrier. In some embodiments, the patient in need of treatment has been diagnosed with cancer.

[0104] In some embodiments, the fusion protein or composition is administered in combination with a T cell therapy, a cancer vaccine, a chemotherapeutic agent, or an immune checkpoint inhibitor (ICI). In some embodiments, the chemotherapeutic agent is a kinase inhibitor, an antimetabolite, a cytotoxin or cytostatic agent, an antihormonal agent, a platinum-based chemotherapeutic agent, a methyltransferase inhibitor, an antibody, or an anticancer peptide. In some embodiments, immune checkpoint inhibitors target PD-L1, PD-1, CTLA-4, CEACAM, LAIR1, CD160, 2B4, CD80, CD86, CD276, VTCN1, HVEM, KIR, A2AR, MHC class I, MHC class II, GALS, adenosine, TGFR, OX40, CD137, CD40, IDO, CSF1R, TIM-3, BTLA, VISTA, LAG-3, TIGIT, IDO, MICA / B, LILRB4, SIGLEC-15, or arginase, and include, but are not limited to, inhibitors of PD-1 (e.g., anti-PD-1 antibodies), inhibitors of PD-L1 (e.g., anti-PD-L1 antibodies), or inhibitors of CTLA-4 (e.g., anti-CTLA-4 antibodies). Examples of T cell therapy include, but are not limited to, CD4+ or CD8+ T cell-based therapy, adoptive T cell therapy, chimeric antigen receptor (CAR)-based T cell therapy, tumor infiltrating lymphocyte (TIL)-based therapy, autologous T cell therapy, and allogeneic T cell therapy. Exemplary cancer vaccines include, but are not limited to, dendritic cell vaccines, vaccines comprising one or more polynucleotides encoding one or more cancer antigens, and vaccines comprising one or more cancer antigen peptides.

[0105] In some embodiments, the fusion proteins of the present disclosure are part of a pharmaceutical composition that includes, for example, the fusion protein and one or more pharma- ceutical acceptable carriers. The pharmaceutical compositions and formulations described herein can be prepared by mixing an active ingredient (e.g., a fusion protein) having a desired degree of purity with one or more optional pharma- ceutical acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), followed by lyophilization. It can be prepared in the form of a dry formulation or an aqueous solution. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations used, and include, but are not limited to, buffers such as phosphate, citric acid and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids; monosaccharides, disaccharides and other carbohydrates such as glucose, mannose or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG). In some embodiments, the fusion protein of the present disclosure is lyophilized.

[0106] Enumerated Embodiments The following enumerated embodiments are representative of some aspects of the present invention. 1. A fusion protein comprising two parts: i) the first portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer, where VH is the variable heavy chain and CH2-CH3 is the Fc domain, an antibody light chain VL-CL, where VL is the variable light chain and CL is the constant light chain, and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; A fusion protein wherein both the first and second moieties bind to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1. 2. A fusion protein comprising two parts, i) the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer, where CH2-CH3 is an Fc domain, and a mutant IL-2 polypeptide, wherein the N-terminus of the mutant IL-2 polypeptide is fused to the C-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; The second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1. Fusion proteins. 3. A fusion protein comprising two parts, i) the first portion is a polypeptide comprising an antibody hinge-CH2-CH3 monomer (wherein CH2-CH3 is an Fc domain) and a mutant IL-2 polypeptide, wherein the C-terminus of the mutant IL-2 polypeptide is fused to the N-terminus of the Fc domain via a linker; ii) the second portion is a polypeptide comprising an antibody heavy chain VH-CH1-hinge-CH2-CH3 monomer and an antibody light chain VL-CL; A fusion protein in which the second portion binds to an epitope on an antigen selected from the following group: human CD8α, human CD8β, and human PD1. 4. The fusion protein of any one of embodiments 1-3, wherein said mutant IL-2 polypeptide exhibits a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having an amino acid sequence depicted in FIG. 1B compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence depicted in FIG. 1A, and a 50% or greater reduction in binding affinity to an IL-2Rα polypeptide having an amino acid sequence depicted in FIG. 1C compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence depicted in FIG. 1A. 5. The fusion protein of embodiment 4, wherein said mutant IL-2 polypeptide further exhibits a 50% or greater reduction in binding affinity to an IL-2Rγ polypeptide having the amino acid sequence depicted in FIG. 1D compared to the binding affinity of a wild-type IL-2 polypeptide having the amino acid sequence depicted in FIG. 1A. 6. The fusion protein of embodiment 4 or 5, wherein said mutant IL-2 polypeptide has two or more amino acid substitutions relative to the wild-type IL-2 amino acid sequence as depicted in FIG. 2 and selected from the group of Q11, E15, H16, L18, L19, D20, Q22, R38, F42, K43, Y45, E62, P65, E68, V69, L72, N88, V91, I92, T123, Q126, S127, I129, S130. 7. The fusion protein of embodiment 6, wherein said mutant IL-2 polypeptide further comprises the amino acid mutation C125A to improve its biophysical properties compared to wild-type IL-2. 8. The fusion protein of embodiment 6 or 7, wherein said first and second Fc domains contain the following Fc mutations, according to EU numbering, to reduce effector function: L234A, L235A, G237A and K322A. 9. The fusion protein of embodiment 6 or 7, wherein said first and second Fc domains contain the following amino acid substitutions: Y349C / T366W (knob) and S354C, T366S, L368A and Y407V (hole) to promote heterodimer formation. 10. One or more isolated polynucleotides encoding the mutant IL-2 polypeptide or fusion protein of any one of embodiments 1-9. 11. One or more vectors, particularly expression vectors, comprising the polynucleotide of embodiment 10. 12. A host cell comprising the polynucleotide of embodiment 10. 13. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 1 to 9 and a pharma- ceutically acceptable carrier. 14. The fusion protein of any one of embodiments 1 to 9 for use as a medicament. 15. A method for treating cancer or a chronic infection comprising administering to a patient a composition according to any of embodiments 1-9 and 13. 16. A method of treating cancer comprising administering to a patient a composition according to any of embodiments 1-9 and 13 in combination with T cell therapy or a cancer vaccine. EXAMPLES

[0107] Example 1 Recombinant DNA techniques Techniques involving recombinant DNA manipulation have been previously described by Sambrook et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989. All reagents were used according to the manufacturer's instructions. DNA sequences were determined by double-stranded sequencing.

[0108] Gene synthesis Desired gene segments were generated by PCR using appropriate templates or synthesized from synthetic oligonucleotides at Thermo Scientific (Pleasanton, CA), ATUM (Newark, CA), Genewiz (South Plainfield, NJ), or GeneScript (Piscataway, NJ). Gene segments flanked by designed restriction endonuclease cleavage sites were digested and then cloned into their respective expression vectors. DNA was purified from transformed bacteria and concentrations were determined by UV-visible spectroscopy. DNA sequencing was used to confirm the DNA sequences of the subcloned gene fragments.

[0109] Isolation of antibody genes Antibodies that bind to CD8 or PD1 antigens were generated using either an in vitro display system or in vivo immunization. For the in vitro display method, non-immune human antibody phage libraries were panned for 5-6 rounds to isolate antibodies against the target antigen. After panning, individual phage clones that showed more specific binding to the target antigen than to non-specific antigens were identified by ELISA. DNA fragments of the heavy and light chain V domains of the specific binders were then cloned and sequenced. Meanwhile, antibodies were also generated from immunization of mice and llamas with recombinant forms of the antigens. From mouse immunization, antibodies were isolated using hybridoma techniques. Briefly, after immunization, B cells from the spleen and / or lymph nodes were fused with myeloma cell lines to generate hybridoma cells. The hybridoma clones were then individually screened using ELISA to identify clones expressing antibodies specific to the antigens. Finally, DNA fragments of the heavy and light chain V domains of the antibodies were cloned from the specific hybridomas and then sequenced. For llama immunization, antibody genes were cloned from peripheral B cells and ligated into phagemid vectors to generate a phage display antibody library. Antibodies were then isolated by panning the phage library against the antigen of interest. After panning, individual phage clones that showed more specific binding to the target antigen than to a non-specific antigen were identified using ELISA. DNA fragments of the heavy and light chain V domains of the specific binders were then cloned and sequenced. The non-human (mouse and llama) derived antibodies were then humanized to remove non-human framework and complementarity determining region mutations.

[0110] Cloning of fusion constructs General information on the nucleotide sequences of human immunoglobulin light and heavy chains can be found at Lefranc IMGT®, the international ImMunoGeneTics information system® 25 years on. Nucleic Acids Res. 2015 Jan;43 The heavy and light chain V domains were obtained from the International ImMunoGeneTics information system (GT®). Amplified DNA fragments of the heavy and light chain V domains were inserted in frame into a human IgG1-containing mammalian expression vector. The IL-2 portion of the construct was cloned in frame with the heavy chain using a (G4S)3 15-mer linker between the C-terminus of the IgG heavy chain and the N-terminus of IL-2. After fusing the IL-2 portion, the C-terminal lysine residue of the IgG heavy chain was removed. To generate a construct with a single IL-2 gene fused to a complete IgG, it was necessary to construct and transfect two heavy chain plasmids for heterodimerization facilitated by knob-into-hole modifications in the IgG CH3 domain. The "hole" heavy chain connected to the IL-2 moiety carried Y349C, T366S, L368A and Y407V mutations in the CH3 domain, while the unfused "knob" heavy chain carried S354C and T366W mutations in the CH3 domain (EU numbering). To abolish FcγR binding / effector function and prevent FcR coactivation, the following mutations were introduced in the CH2 domain of each of the IgG heavy chains: L234A / L235A / G237A (EU numbering). Expression of the antibody-IL-2 fusion constructs was driven by a CMV promoter and transcription was terminated by a synthetic polyA signal sequence located downstream of the coding sequence.

[0111] Preparation of fusion proteins containing IL-2 polypeptides Constructs encoding fusion proteins with IL-2 polypeptides as used in the examples were produced by co-transfection of exponentially growing Expi293 cells with mammalian expression vectors using polyethyleneimine (PEI). Briefly, IL-2 fusion constructs were first purified by affinity chromatography using a protein A matrix. The protein A column was equilibrated in phosphate buffered saline (PBS) and washed. The fusion constructs were eluted with 20 mM sodium citrate, 50 mM sodium chloride, pH 3.6. The elution fractions were pooled and dialyzed into 10 mM MES, 25 mM sodium chloride pH 6. The proteins were further purified using ion exchange chromatography (Mono-S, GE Healthcare) to purify the heterodimer over the homodimer. After loading the protein, the column is washed with 10 mM MES, 25 mM sodium chloride pH 6. The protein was then eluted with an increasing gradient of sodium chloride from 25 mM to 500 mM in 10 mM MES pH 6 buffer. The main eluent peak corresponding to the heterodimer was collected and concentrated, and the purified protein was then refined by size-exclusion chromatography (Superdex 200, GE Healthcare) in PBS.

[0112] Protein concentrations of purified IL-2 fusion constructs were determined by measuring the optical density (OD) at 280 nm using the molar extinction coefficient calculated based on the amino acid sequence. Purity, integrity and monomeric state of the fusion constructs were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1,4-dithiothreitol) and stained with Coomassie Blue (SimpleBlue™ SafeStain, Invitrogen). The NuPAGE® Pre-Cast Gel System (Invitrogen) was used according to the manufacturer's instructions (4–20% Tris-glycine gels or 3–12% Bis-Tris). Immunoconjugate samples were analyzed for aggregate content using a Superdex 200 10 / 300 GL analytical size-exclusion column (GE Healthcare).

[0113] pSTAT5 and Ki-67 assays to measure selective activation of antigen-expressing blood immune cells The activity of IL-2 fusion proteins was determined in an assay in human peripheral blood mononuclear cells (PBMCs) measuring the phosphorylation of STAT5. PBMCs were isolated from blood of healthy donors using Ficoll-Paque Plus (GE Healthcare) and red blood cells were lysed using ACK lysis buffer (Gibco) according to the manufacturer's instructions. Typically, PBMCs were cultured in serum-free RPMI 1640 medium at 2 × 10 6 Cells were resuspended at 100 cells / ml and aliquoted into 96-well U-bottom plates (50 μl per well). IL-2 fusion proteins and control proteins, e.g., recombinant human IL-2 and control (targeted to HA) fusion proteins, were diluted to the desired concentrations and added to the wells (50 μl was added as 2× stimulation). Incubations were typically performed for 30 min at 37° C., after which they were stopped with 100 μl of pre-warmed 4% PFA (2% final) for 10 min at 37° C. Cells were then stained with antibodies against the following surface markers: CD45 (clone HI30), CD3 (UCHT1, BD Biosciences), CD8α (SK1, Biolegend; RPA-T8, Biolegend), CD4 (RPA-T4, Biolegend), and CD25 (M-A251, Biolegend). The cells were washed twice with washing buffer (2% FBS in PBS) and fixed with 4% PFA for 10 min at room temperature. After fixation, the cells were fixed with pre-cooled Phosflow Perm buffer III (BD Cells were permeabilized using a 30-mL ELISA kit (BD Biosciences) according to the manufacturer's protocol. After permeabilization, cells were stained with antibodies against intracellular markers (pSTAT5 [pY694], clone 47, BD Biosciences, and / or perforin, clone δG9, BD Biosciences) and analyzed by flow cytometry. Data are expressed as percent pSTAT5 positive and in some cases as pSTAT5 mean fluorescence intensity (MFI) and imported into GraphPad Prism to determine ECs for each construct. 50value was determined.

[0114] To measure cellular changes induced by IL-2 fusion proteins further downstream from pSTAT5, such as proliferation, a flow cytometry assay was used to detect expression of the intracellular proliferation marker Ki-67. Briefly, PBMCs were isolated as described above and incubated in serum-supplemented RPMI1640 (10% FBS) or serum-free AIM V medium (Gibco) for 4–6 days in the presence of IL-2 fusion proteins and controls. Staining for Ki-67 (clone Ki-67, Biolegend) was performed using Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocol. Data were expressed as Ki-67 percent positive and imported into GraphPad Prism to determine ECs for each construct when possible. 50 value was determined.

[0115] pSTAT5 and Ki67 assays for measuring activation of mouse immune cells Splenocytes were isolated from the spleens of B6 mice by placing the spleens onto a 70 mm strainer and using a plunger to wash the cells through the strainer with PBS. Red blood cells were lysed with ACK lysis buffer and cells were diluted to 20 × 10 per ml of RPMI medium. 6The cells were resuspended in 75 ml of PBS-2% FBS and incubated for 1 h at 4°C. The cells were washed 3 times with PBS-2% FBS and stained in 50 μl of FACS buffer containing antibodies against CD3 (17A2), CD4 (GK1.5), CD8a (53-6.7), CD8b (YTS156.7.7), CD25 (7D4), and pSTAT5 (clone 47). The samples were washed twice and analyzed by flow cytometry. For Ki67 assay, 1 x 10 5 Splenocytes were seeded in RPMI medium supplemented with 10% FBS in 96-well U-bottom plates and cultured at 37°C for 5 days before staining for Ki-67. Briefly, cells were surface stained with antibodies against CD3 (145-2C11), CD4 (GK1.4), CD8 (53-6.7), CD25 (PC61) and NK1.1 (PK136), then fixed and permeabilized using Foxp3 / Transcription Factor Staining Buffer Set (Thermo Fisher Scientific) according to the manufacturer's protocol. Ki67 antibody (clone 16A8) was added for 45 min at 4°C, after which cells were washed and analyzed by flow cytometry.

[0116] Determination of binding affinity by surface plasmon resonance (SPR) for IL-2Rα and IL-2Rβ Kinetic rate constants of IL-2 fusion proteins for human and cynomolgus monkey CD8α, CD8β, and PD1 antigens, and for human IL-2Rα and IL-2Rβ rate constants) (kon and koff) and affinity (K D) was measured by surface plasmon resonance (SPR) at 37 °C using a BIAcore (GE Healthcare) (10 mM phosphate, 150 mM sodium chloride pH 7.4, 0.005% Tween 20). Briefly, to determine affinity, IL-2 fusion proteins were captured via their Fc onto a CM4 sensor chip by a covalently immobilized anti-human Fc capture antibody at 0.75 μg / mL and a flow rate of 10 μL / min for 30 s. No antibody was captured on flow cell 1 to serve as a reference surface. Various concentrations of antigen (1 mM to 1.4 nM in 3-fold dilutions) or IL-2 receptor (3 mM to 12 nM in 3-fold dilutions) were injected as test substances over the captured IL-2 fusion proteins. The association phase was recorded for 120 s and the dissociation phase was recorded for 600 s. Sensorgrams were double-referenced and globally fitted to a 1:1 Langmuir mass transfer model to determine association rates (k), dissociation rates (k), and equilibrium dissociation constants (K = k / k) using Biacore Evaluation Software.

[0117] In an alternative format of the assay, antigen was first captured on the chip and then IL-2 fusion protein was injected as the test substance. In this case, histidine-tagged IL-2 receptor, either generated in-house or purchased commercially, was diluted to 0.125 μg / mL in running buffer and captured for 1 min at 10 μL / min on a CM4 sensor chip amine-coupled with anti-HIS antibody. No IL-2 receptor was captured on flow cell 1 to serve as a reference surface. IL-2 fusion protein at 1000 nM to 4.1 nM (3-fold dilution series) was injected on flow cells 1 and 2 for 2 min and allowed to dissociate for 1 min. The surface was regenerated between analysis cycles with two 30 s injections of 10 mM glycine pH 1.7. Sensorgrams were double-referenced and globally fitted to 1:1 Langmuir to determine k, k and K using Biacore Evaluation Software.

[0118] Example 2: Ability of IL-2 and IL-2 variants to activate splenocyte subsets This example describes experiments to test the ability of IL-2 fusion compounds to activate STAT5 in mouse NK cells and induce NK-mediated toxicity in mice.

[0119] The ability of IL-2 and IL-2 variants with reduced binding to CD25 / IL2Rα to activate spleen cell subsets was tested in a STAT5 assay. IL-2 and a previously published IL-2 variant (IL-2v) fused to a control antibody (xHA), xHA-IL-2v, were used to stimulate mouse splenocytes containing CD8 T cells, CD4 T cells and NK cells. STAT5 activation in different spleen subsets was measured by flow cytometry as described in Example 1. CD8 T cells were identified with the CD3+CD4- gate / subset, Treg cells were identified as CD3+CD4+CD25+, and NK cells were identified as CD3-CD49b+.

[0120] Figure 5 shows the results of this experiment. NK cells were about 10-fold more sensitive to IL-2 stimulation than CD8+ T cells, with Treg cells being the most sensitive (left side of Figure 5). In the case of IL-2v, due to their reduced binding to CD25, NK cells were about 10-fold more sensitive to IL-2v stimulation than both CD8 T cells and Treg cells (right side of Figure 5).

[0121] To test NK cell-induced toxicity, we measured weight loss in mice treated with IL-2 variants with reduced binding to CD25 / IL2Ra. NK cell-induced toxicity from treatment with IL-2 variants can be manifested as weight loss. For this experiment, 8-10 week old B6 mice were subcutaneously injected with a single dose of the indicated compound and their weights were recorded daily. xHA-IL-2v was administered at 1 mg / kg or 5 mg / kg together with anti-PD1 (xPD1) at 2.5 mg / kg, whereas TAg-IL-2v was administered alone at 5 mg / kg. NK cells were depleted with anti-NK1.1 antibody (PK136 clone) at 200 mg / mouse ip. The depleting antibody was injected 2 days before and 1 day after TAg-IL-2v administration to maintain depletion.

[0122] Figures 6A and 6B show the results of these experiments. IL-2 variants (IL-2v) fused to control xHA antibodies (xHA-IL-2v) or FAP antibodies (TAg-IL-2v) induced weight loss in mice (Figure 6A). This weight loss was mediated by NK cells, as evident in mice in which NK cells were depleted with NK1.1 antibodies (Figure 6B). Such toxicity mediated by NK cells may limit the maximum tolerated dose of IL-2-based therapeutics in humans. The maximum tolerated dose for control antibody-targeted IL2v or tumor antigen-targeted IL2v in mice was well below 5 mg / kg.

[0123] Example 3: Characterization of anti-mouse CD8 antibodies This example describes the characterization of anti-mouse CD8 antibodies.

[0124] result The binding affinity of anti-mouse CD8 antibodies was determined by flow cytometry analysis. Fresh splenocytes were incubated with either xCD8ab1 (clone 2.43), xCD8ab2 (clone YTS156.7.7) or xCD8ab2.1 for 2 h at 4 °C. The xCD8ab1 (clone 2.43) and xCD8ab2 (clone YTS156.7.7) sequences have been published previously. The xCD8ab2.1 clone was derived from xCD8ab2 by introduction of the mutations N95A (VH) and D92A (VK). After incubation with anti-mouse CD8 antibodies, cells were stained with antibodies against CD3, CD4, CD8 and anti-hFc (HP6017, Biolegend), the last of which was used to measure the binding of a CD8-IL2 fusion containing hFc. Stained cells were washed and analyzed by flow cytometry, and mean fluorescence intensity (MFI) of staining with anti-hFc was used to display binding. As shown in Figure 7, xCD8ab2 had higher affinity for CD8+ T cells than xCD8ab1. xCD8ab2.1 is a lower affinity variant of xCD8ab2 that was generated by introducing two mutations (N95A (VH) and D92A (VK)) into xCD8ab2.

[0125] The MHC blocking status of anti-mouse CD8 antibodies was also tested. Binding of certain CD8 antibodies can inhibit T cell activation by interfering with the interaction of CD8 molecules on T cells with MHC molecules on antigen-presenting cells or tumor cells. For this experiment, CD8+ T cells were purified from splenocytes from OT-I transgenic mice and co-cultured with EL-4-OVA cancer line (ATCC) at 100,000 cells each for 24 hours. Cells were analyzed by cell surface staining and flow cytometry for upregulation of activation markers such as CD25 and CD69. As shown in Figure 8, both xCD8ab1 and xCD8ab2 blocked T cell activation. This suggests that these antibodies interfered with and blocked the interaction of CD8 with MHC. xCD8ab2 blocked T cell activation more potently, correlating with its higher binding affinity to CD8.

[0126] Example 4: Characterization of IL-2 muteins fused to anti-CD8 antibodies This example describes the characterization of an IL-2 mutein fused to a CD8 antibody.

[0127] method B16 Mouse Tumor Model C57BL6 female mice (Jackson Labs) aged 8–10 weeks were housed and acclimated in an animal facility. Cultured B16.F10 cells (ATCC, CRL-6475) were harvested and resuspended in serum-free medium (1x DMEM, Sigma D6429) at 5 × 106 cells / mL for implantation. Mice were shaved and cultured at 5 × 10 5 Cells (100 μL) were implanted subcutaneously in the upper rear of the hind leg. Tumors were measured until they reached a tumor volume of 60-120 mm3 (TV = width x width x length x 0.5) approximately 8 days after implantation. Mice were then randomly assigned to groups by tumor volume. Each mouse was weighed and dosed subcutaneously under the nape of the neck. Tumor volumes and body weights were monitored until the end of the study (30-40 days after the first dose) or until the maximum tumor volume (2000 mm3). 3At the end of the study, mice were euthanized with CO2 and an appropriate secondary euthanasia agent.

[0128] CT26 mouse tumor model BALB / c female mice were shaved and transfected with 2 × 10 5 The cells (100 μL) were subcutaneously transplanted into the upper hind leg. The tumor volume was 60–120 mm approximately 8 days after transplantation. 3 Tumor volumes were measured (TV=width×width×length×0.5) until the tumor reached a volume of 100 μg / kg. Each mouse was then weighed and dosed subcutaneously with the indicated compound (9 mice per group).

[0129] Analysis of CD8+ T cells in blood and tumors of B16 tumor-bearing mice B6 mice were injected with B16 tumor cells, and tumors grew to 200–250 mm 3 After growth to 30 min, mice were administered the indicated IL-2 fusions at 1 mg / kg. Tumors were removed 5 days after administration, digested to single cells, and profiled by flow cytometry to detect CD8+ T cells and NK cells (NK1.1+CD3-). Briefly, tumors were digested in Miltenyi Gentle MACS C tubes using the Mouse Tumor Dissociation Kit (Miltenyi Biotec, 130-096-730) according to the manufacturer's protocol. Isolated cells were counted and collected at 10 × 10 6 Cells were stained with antibodies against CD45, CD3, CD4, CD8, CD25 and CD49b. Blood was also collected from mice (50 μl), lysed with ACK lysis buffer, washed with wash buffer (PBS / 0.5% BSA / 2 mM EDTA) and stained with lineage markers to identify CD8+ T cells and NK cells. Cells were analyzed by flow cytometer.

[0130] result IL-2 mutein variants fused to CD8 antibodies were tested for selective targeting of CD8+ T cells compared to other immune cells expressing IL-2R. IL-2 muteins were fused to a previously published anti-mouse CD8 antibody, xmCD8ab1 (2.43 clone), in format B. Mouse splenocytes were treated with IL-2 mutein fusions and STAT5 assays were performed as described in Example 1. Table 1 and Figure 9 summarize the results of this experiment.

[0131] IL-2 muteins fused to CD8 antibodies in format B selectively targeted CD8+ T cells over other cells expressing IL-2R, including NK cells. Because Tregs express CD25 but other cells do not, activity against Tregs was used as a surrogate for IL-2Rα / CD25 binding. Other sequences also targeted CD8 T cells preferentially over NK cells, but had increased activity against Tregs as a result of higher CD25 binding. All of the sequences included in Table 1 showed at least a 50% reduction in binding to IL2Rα / CD25, as the activity of IL-2 in this assay was <0.001 nM (Figure 5, panel A). Additionally, several sequences (m3, m4, m5, m10 as shown in Table 1) were identified that had the lowest activity against Tregs. [Table 1]

[0132] The efficacy of CD8-IL-2 and TAg-IL-2v in combination with anti-PD1 was tested in a B16 tumor model. IL-2m10 mutant proteins fused to one of two different CD8 antibodies, xmCD8ab1 (2.43 clone) and xmCD8ab2 (YTS1567.7), in format C, were administered as shown in mice implanted with B16 tumors. As shown in Figure 10, anti-PD1 antibodies showed only moderate efficacy in this model, slowing tumor growth but not resulting in complete response or cure of any mice. CD8-IL2 mutant proteins induced more complete tumor regression when administered in combination with anti-PD1 than TAg-IL-2v in a single dose regimen. CD8ab2 fusions induced more complete tumor regression than CD8ab1 fusions.

[0133] The effect of treatment with CD8-IL-2 fusions on CD8+ T cell accumulation in blood and tumors was examined using a B16 tumor model. Mice implanted with B16 tumor cells were administered IL-2 fusions at 1 mg / kg together with xPD1 at 5 mg / kg. CD8+ T cell levels were measured by flow cytometry in both tumor and blood samples. As shown in Figures 11A and 11B, CD8-IL2 induced more CD8 T cell expansion than TAg-IL-2v in blood (Figure 11A), while TAg-IL-2v induced more NK cell expansion. Similarly, a significant expansion of CD8+ T cells was observed (about 17-fold) in tumors in mice administered CD8-IL2, whereas only about 3-fold CD8+ T cell expansion was observed in the presence of TAg-IL-2v (Figure 11B).

[0134] To compare the performance of CD8-IL-2 and TAg-IL-2v, tumor regression was assessed in mice implanted with CT26 tumor cells and treated with either CD8-IL-2 alone or TAg-IL-2v alone. As shown in Figures 12A-12C, IL-2m4 mutein fused to xmCD8ab2 in format C induced more complete and partial tumor regression than TAg-IL-2v in the CT26 colon tumor model when both drugs were administered as single agents.

[0135] The effect of CD8 antibody affinity on potency was tested for fusions with IL-2m4. For this experiment, the IL-2 mutein IL-2m4 was fused to either xmCD8ab2 or xmCD8ab2.1 in format C. xmCD8ab2.1 is a lower affinity version of xmCD8ab2. STAT5 assays were performed on mouse splenocytes as described in Example 1. As shown in Figure 13, xmCD8ab2.1-IL-2m4 fusions had lower potency and lower selectivity for CD8+ T cells than NK cells compared to xmCD8ab2-IL-2m4.

[0136] xmCD8ab2-IL2m4 and xmCD8ab2.1-IL2m4 were also tested for their ability to expand CD8+ T cells in vivo. Blood was collected from naive B6 mice treated with the indicated compounds at 1 mg / kg. Levels of NK cells and CD8+ T cells were determined by flow cytometry. As shown in FIG. 14, both fusions induced a higher expansion of CD8 T cells than NK cells in vivo.

[0137] Example 5: Characterization of anti-CD8:IL-2 mutein fusion proteins with reduced binding to IL-2Rβ and IL-2Rγ This example describes the characterization of a xmCD8-IL-2 mutein that has reduced binding to IL-2Rβ / γ.

[0138] method STAT5 assay Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins with anti-CD49b staining antibody in RPMI medium for 30 minutes, after which the cells were stained for cell surface markers (CD3, CD4, CD8, CD25) and for intracellular STAT5 according to the protocol in Example 1. Data for STAT5 in the indicated cell subsets are expressed as mean fluorescence intensity (MFI).

[0139] Ki67 assay Splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins for 5 days in complete RPMI medium, after which cells were stained for cell surface markers (CD3, CD4, CD8, CD25, NK1.1) and for intracellular markers of proliferation, Ki67, IL-2Rβ / γ and downstream signaling events from STAT5 according to the protocol in Example 1.

[0140] result Given that the active dose of xmCD8ab2-IL2m4 induced detectable NK cell expansion, additional mutations were introduced into IL2m4 to further reduce its activity against IL2R+ cells, including NK cells. Mutations at the IL-2 interaction surface of IL-2Rβ / γ (predicted to reduce binding to IL-2Rbg) were selected. IL-2 muteins with selected mutations that prevent binding to IL-2R were fused to xmCD8ab2 antibody in format C and tested in STAT5 assays using mouse splenocytes. Table 2 and Figures 15A and 15B show the results of these experiments. Table 2 shows the list of mutations. [Table 2]

[0141] Fusions between IL-2 muteins with selected mutations and the xmCD8ab2.1 antibody in format C were also generated and tested in a STAT5 assay using mouse splenocytes. Table 3 and Figures 16A and 16B show the results of this experiment. [Table 3]

[0142] IL-2 muteins with selected mutations were fused to xmCD8ab2 antibody in format C and tested in Ki67 assay using mouse splenocytes. Ki67 is an intracellular marker of proliferation and represents downstream signaling events of IL-2Rβ / γ and STAT5. Table 4 and Figures 17A and 17B summarize the results of these experiments. TAg-2v fusion in format B was included as a reference. [Table 4]

[0143] IL-2 muteins with selected mutations listed in the table below were fused to the xmCD8ab2.1 antibody in format C and tested in the Ki67 assay using mouse splenocytes. Table 5 and Figures 18A and 18B show the results of these experiments. [Table 5]

[0144] The potency for CD8 T cells and NK cells for representative molecules is summarized in Figure 19. The CD8-IL2 fusions generated had a range of selectivities for CD8 T cells over NK cells, with xmCD8ab2-IL2m4 and xmCD8ab2-IL2m4.2 being the most selective (>1000 fold), followed by xmCD8ab2.1-IL2m4 (~50-100 fold), and xmCD8ab2.1-IL2m4.2 being the least selective (~10 fold). The m4 mutein fusion had reduced binding to IL-2Rα, while the m4.1 and m4.2 muteins contained additional mutations that reduced binding to IL-2Rβγ.

[0145] Example 6: Testing the Effect of CD8:IL2 Fusion Protein in Combination with Anti-PD-1 in the B16 Tumor Model result Four representative CD8-IL2 fusions with varying degrees of selectivity for CD8 T cells compared to NK cells were tested in the B16 tumor model as described in Example 4. All mice were administered 1 mg / kg of the indicated fusion together with 5 mg / kg of anti-PD1. As shown in Figure 20, CD8-IL-2 performed better than TAg-IL-2v (see Figure 10) at the lower dose. The CD8-IL2 fusion with the lowest selectivity for CD8 T cells had the least efficacy in the B16 model, approaching that observed for TAg-IL-2v (Figure 10). A >10-fold selectivity was required for the highest efficacy and >40% tumor-free mice.

[0146] The efficacy of CD8-IL-2 was further tested by analyzing the expansion of tumor antigen-specific T cells upon treatment. B6 mice were injected with B16 tumor cells, and tumors were grown to 200–250 mm 3After growth to 30 min, the mice were administered the indicated IL-2 fusions at 1 mg / kg together with xPD1 at 5 mg / kg. Tumors were removed 5 days after administration, digested to single cells, and profiled by flow cytometry to detect CD8+ T cells and NK cells (NK1.1+CD3-). Cells were also stained with p15E tetramer (TB-M507-2, MBL) according to the manufacturer's protocol to detect T cells that recognize the p15E tumor antigen. As shown in Figure 21, both xmCD8ab2-IL2m4.2 and xmCD8ab2.1-IL2m4 induced a >15-fold expansion of total intratumoral CD8+ T cells, and a 5- to 17-fold expansion of p15E tumor antigen-specific T cells, with low to no expansion of NK cells.

[0147] STAT5 assay was performed to compare the potency of bivalent low affinity fusions and high affinity monovalent fusions. Splenocytes from B6 mice bearing B16 tumors were incubated with fusion proteins for 30 minutes in RPMI medium, after which cells were stained for cell surface markers (CD3, CD4, CD8, CD25) and for intracellular STAT5 according to the protocol in Example 1. As shown in Figure 22, bivalent low affinity fusions had similar potency as high affinity monovalent derivatives. IL-2m4.2 fusions fused to high affinity xmCD8ab2 antibody (in format C) or to bivalent xmCD8ab2.1 antibody (in format A) had similar potency on CD8+ T cells and much more potency than monovalent xmCD8ab2.1-IL2m4 (format C) fusions.

[0148] To further test the affinity of the bivalent C-terminal format (format A), the bivalent xmCD8ab2.1 antibody (in format A) was tested in the B16 tumor model as described in Example 4. Mice were administered PBS as a control or 1 mg / kg of the indicated fusion together with 5 mg / kg of anti-PD1 (9 mice per group). As shown in Figure 23, IL-2m4.2 fusions fused to high affinity xmCD8ab2 antibody (in format C) or to bivalent xmCD8ab2.1 antibody (in format A) had similar efficacy in vivo as IL-2m4.2 fusions fused to high affinity xmCD8ab2 antibody (in format C) (see Figure 20). Thus, the bivalent C-terminal format (format A) is also highly effective.

[0149] Blocking of CD8 T cell activation by CD8 antibodies was also tested. CD8+ T cells were purified from splenocytes from OT-I mice and co-cultured with EL-4-OVA line (ATCC) at 100,000 cells each for 24 hours. Cells were analyzed for upregulation of activation markers such as CD25 and CD69 by cell surface staining and flow cytometry as described in Example 3. As shown in Figure 24, certain CD8 antibodies did not block CD8 T cell activation. xmCD8ab3 antibody did not block CD8 T cell activation even at a concentration of 200 nM. xmCD8ab3 antibody was bivalent.

[0150] Blocking of CD8 T cell activation by CD8 antibodies was also tested. CD8+ T cells were purified from splenocytes from OT-I mice and co-cultured with EL-4-OVA line (ATCC) at 100,000 cells each for 24 hours. Cells were analyzed for upregulation of activation markers such as CD25 and CD69 by cell surface staining and flow cytometry as described in Example 3. As shown in Figure 24, certain CD8 antibodies did not block CD8 T cell activation. xmCD8ab3 antibody did not block CD8 T cell activation even at a concentration of 200 nM. xmCD8ab3 antibody was bivalent.

[0151] To compare the in vivo potency of xmCD8ab2 and xmCD8ab3 fusions, splenocytes from B6 mice bearing B16 tumors were incubated with the indicated proteins for 30 minutes in RPMI medium, after which the cells were stained for cell surface markers (CD3, CD4, CD8, CD25, NK1.1) and for intracellular STAT5 according to the protocol in Example 1. As shown in Figure 25, xmCD8ab2-IL2m4.2 and xmCD8ab3-IL2m4.2 showed similar activity against CD8+ T cells in vitro.

[0152] IL-2m4.2 fused to non-MHC-blocking antibodies was also tested in the B16 tumor model as described in Example 4. Mice were administered PBS, 0.3 mg / kg (Figure 26A) or 1 mg / kg (Figure 26B) of the indicated fusions together with 5 mg / kg anti-PD1 (9 mice per group). As shown in Figures 26A and 26B, the non-MHC-blocking antibodies were more optimal in vivo. IL-2m4.2 fusions fused to non-MHC-blocking xmCD8ab3 antibodies (in format C) were much more effective than IL-2m4.2 fusions fused to MHC-blocking xmCD8ab2 antibodies (in format C).

[0153] Preferential targeting of IL-2 mutant protein fusions to PD1+ T cells, both CD8+ and Treg, was also tested. Size 300-600 mm 3B16 tumors were removed from mice and digested to single cells. CD45+ cells were purified (Miltenyi LS columns, according to manufacturer's protocol) and stimulated with the indicated fusion proteins for 30 min. Cells were stained for cell surface markers (CD3, CD4, CD8, CD25, CD49b, and PD1) and for intracellular phospho-STAT5. As shown in Figure 27, fusions of the IL-2 mutein IL2m10 and anti-PD1 antibody preferentially targeted PD1+ T cells over PD1- T cells, but targeted both CD8+PD1+ T cells and CD4+CD25+PD1+ Treg cells.

[0154] Example 7: Effect of IL-2 mutations on the activity and selectivity of CD8-IL-2 fusions in hPBMCs This example describes the effect of IL-2 mutations on the activity and selectivity of CD8-IL-2 fusions in hPBMCs.

[0155] result To characterize the effect of IL-2 mutations on the activity of CD8-IL-2 fusions in hPBMCs, the indicated IL-2 mutant proteins were fused to the xmCD8ab1 antibody and tested in a STAT5 assay with hPBMCs. The xmCD8ab1 antibody did not recognize human CD8. The resulting data was used to rank each mutation according to its effect on STAT5 activity. Tables 6, 7 and 8 summarize the results of this experiment. Tregs are shown as a representative example of cells expressing IL-2Rβγ.

[0156] Table 6 depicts the activity of the IL-2 muteins IL-2m1 to IL-2m10 fused to xmCD8ab1, all in format B, against human Tregs, comparable to IL-2v fused to the control xHA antibody. Table 6 shows that the IL-2 muteins IL-2m1 to IL-2m10 fused to xmCD8ab1 all have significantly reduced activity against Tregs compared to wild type IL-2, resulting in significantly reduced binding to IL-2Ra, making them comparable to IL-2v, which has no binding to IL-2Ra (Klein et al., 2003). et al, Oncoimmunol. 2017; 6(3); e1277306). [Table 6]

[0157] Table 7 depicts the activity of IL-2 muteins IL-2m10.1 to IL-2m10.11 fused to xmCD8ab1 against human Tregs compared to IL-2m10 fused to xmCD8ab1, all in format B. Table 7 shows that IL-2m10.1 to IL-2m10.11 fused to xmCD8ab1 all have significantly reduced activity against Tregs compared to IL-2m10. Since IL-2m10 has reduced or no activity against IL2Ra, the additional mutations present in IL-2m10.1 to IL-2m10.11 reduced the activity of the molecules by reducing binding to IL-2Rβγ. [Table 7]

[0158] Table 8 depicts the activity of IL-2 mutein IL-2m4.1 to IL-2m4.6 and IL-2m4.9 to IL-2m4.24 fused to xmCD8ab1 against human Tregs compared to IL-2m4 fused to xmCD8ab1, all in format B. Since IL-2m4 has reduced or no activity against IL2Rα, the additional mutations present in the IL-2 mutein fusion proteins shown in Table 8 reduced the activity of the molecule by reducing binding to IL-2Rβγ. Reduced activity against Tregs, and therefore reduced binding to IL-2Rbg, was observed for all of the IL-2 mutein fusions, except for IL-2m4.9, IL-2m4.10, IL-2m4.12 and IL-2m4.16. [Table 8]

[0159] Selective stimulation of human CD8+ T cells by CD8-IL-2 mutein fusions was also tested. The indicated IL-2 muteins were fused to the previously published anti-human CD8 antibody clone OKT8 (xhCD8ab) in format C and tested in the STAT5 assay with hPBMCs. Tables 9 and 10 summarize the results of this experiment. CD8-IL2 mutein fusions selectively and potently stimulated human CD8 T cells. Since IL-2m4 has reduced or no activity against IL2Ra, additional mutations present in the IL-2 mutein fusion proteins shown in Table 9 reduced the activity of the molecule by reducing binding to IL-2Rbg. Reduced activity against Tregs and therefore reduced binding to IL-2Rbg was observed for all of the IL-2 mutein fusions except for IL-2m4.26, IL-2m4.27, IL-2m4.28 and IL-2m4.29.

[0160] Certain mutations were not useful because they resulted in very weak CD8 T cell activation (i.e., an IL-2 mutein containing the N88E mutation only weakly activated CD8 T cells in the context of a fusion with xhCD8ab, whereas N88A / S / T ​​generated a CD8-IL2 molecule with potent activity against CD8 T cells). [Table 9] [Table 10]

[0161] Tables 11 and 12 depict the activity of IL-2m10.12 muteins with the Q126A mutation in the context of fusions to either the control antibody (xHA) or the xhCD8ab antibody. This mutation in the context of a CD8-IL2 fusion reduced the activity of the CD8-IL2 fusion on human Tregs compared to those containing the IL-2m10 mutein (Tables 6 and 7), but allowed potent selective activation of CD8+ T cells.

Table 11

Table 12

Claims

[Claim 1] The invention described in the specification.

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