Activatable cytokine polypeptides and methods for using them

Abstract

The present disclosure features fusion proteins that are conditionally active variants of a cytokine of interest. In one aspect, the full-length polypeptides of the present invention have reduced or minimal cytokine receptor activating activity despite containing a functional cytokine polypeptide. When activated, for example, by cleavage of a linker connecting a blocking moiety, e.g., a steric blocking polypeptide, to the active cytokine, the cytokine can bind to its receptor and cause signal transduction. Typically, the fusion protein further comprises an in vivo half-life extending element that can be cleaved from the cytokine in the tumor microenvironment.

Description

[Technical Field] 【0001】 This application claims the benefits of U.S. Provisional Application No. 62 / 935,605, filed on November 14, 2019, the entirety of which teachings are incorporated herein by reference. 【0002】 Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy was created on November 12, 2020, is named 761146_000146_SL.txt, and has a size of 2,974,211 bytes. [Background technology] 【0003】 The development of mature, immune-normal lymphoid cells from less specialized precursors, their subsequent antigen-induced immune responses, and the suppression of these unwanted autoreactive responses are largely dependent on and regulated by cytokines (including interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21) that utilize receptors within the common gamma chain (γc) family (Rochman et al., 2009), as well as family members including IL-12, 18, and 23. IL-2 is essential for Treg cell development in the thymus and critically modulates several crucial aspects of mature peripheral Tregs and normal antigen-activated T cells. IL-2 has been studied extensively, partly due to its potent T-cell growth factor activity in vitro, as this activity has presented a potential means of directly enhancing immunity, for example, in cancer and AIDS-HIV patients, or as a target to counter unwanted responses, such as graft rejection and autoimmune diseases. While in vitro studies on IL-2 provided strong theoretical interpretations for these studies, the function of IL-2 in vivo is clearly far more complex, as first demonstrated in IL-2-deficient mice exhibiting rapid, lethal autoimmune syndrome rather than loss of immunity (Sadlack et al., 1993, 1995). Later, similar observations were made when the genes encoding IL-2Rα (Il2ra) and IL-2Rβ (Il2rb) were individually removed (Suzuki et al., 1995; Willerford et al., 1995). 【0004】 This invention relates to conditionally activated and / or targeted cytokines for use in the treatment of cancer and other diseases that depend on the upregulation or downregulation of the immune system. For example, the antitumor activity of several cytokines is well known and described, and some cytokines have already been used therapeutically in humans. Cytokines such as interleukin-2 (IL-2) and interferon-alpha (IFNα) have shown positive antitumor activity in patients with various tumors, such as metastatic renal cell carcinoma, hairy cell leukemia, Kaposi's sarcoma, melanoma, and multiple myeloma. Other cytokines such as IFNβ, tumor necrosis factor (TNF)α, TNFβ, IL-1, 4, 6, 12, 15, and CSF have shown some antitumor activity against several types of tumors and are therefore subjects for further investigation. [Overview of the Initiative] 【0005】 This specification provides therapeutic proteins, nucleic acids encoding such proteins (e.g., DNA, RNA, mRNA), and methods and compositions for using such proteins and nucleic acids for the treatment of diseases or disorders, such as proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, etc. In one embodiment, the fusion protein is one amino acid sequence from SEQ ID NOs: 193 to 271. The specific fusion proteins disclosed herein are designated as ACP200-208, ACP211, ACP213-ACP215, ACP240-ACP245, ACP247, ACP284-ACP292, ACP296-ACP300, ACP302-ACP306, ACP309-ACP314, ACP336-ACP359, ACP371-ACP379, ACP383-ACP434, ACP439-ACP447, or ACP451-ACP471. This disclosure also relates to nucleic acids (e.g., DNA, RNA, mRNA) encoding fusion proteins, methods for producing fusion proteins, compositions comprising fusion proteins, combinations of two or more fusion proteins, and methods for using fusion proteins to treat cancer, including one or more fusion proteins combined with another therapeutic agent. 【0006】 The present invention features fusion proteins that are conditionally active variants of cytokines of interest. Particularly interesting cytokines include IL-2, IL-12, and IFN. In one embodiment, the full-length polypeptide of the present invention has reduced or minimal cytokine receptor activating activity despite containing a functional cytokine polypeptide. Cytokines, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFN alpha, IFN beta, IFN gamma, TNF alpha, lymphotoxin, TGF beta 1, TGF beta 2, TGF beta 3, GM-CSF, CXCL10, CCL19, CCL20, CCL21, or any of the above functional fragments, mutaines, functional variants, or subunits, can bind to their receptors and induce signal transduction when activated, for example, by cleavage of an inhibitory moiety, such as a linker linking a steric inhibitory polypeptide in succession, to an active cytokine. Optionally, the full-length polypeptide may also include an inhibitory polypeptide moiety that provides additional beneficial properties. For example, a full-length polypeptide may contain an inhibitory polypeptide moiety that extends the serum half-life and / or directs the full-length polypeptide to a desired cytokine site of action. Alternatively, a full-length fusion polypeptide may contain a serum half-life extending element and / or a target-directing domain separate from the inhibitory polypeptide moiety. Preferably, the fusion protein contains at least one element or domain that can extend the circulating half-life in vivo. Preferably, this element is enzymatically removed at a desired site in the body (e.g., protease cleavage in the tumor microenvironment) to restore the pharmacokinetic properties of the loaded drug molecule (e.g., IL2, IL-12, IFNb, or IFNa) to substantially the same extent as that of the naturally occurring loaded drug molecule. The fusion protein may target a desired cell or tissue. 【0007】 A fusion polypeptide typically comprises a cytokine polypeptide [A], an inhibitory moiety [D], an optional half-life extension moiety [H], and a protease-cleavable polypeptide linker. The cytokine polypeptide, the inhibitory moiety, and the optional half-life extension element (if present) are functionally linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has a weakened cytokine receptor activating effect; for example, the cytokine receptor activating effect of a fusion polypeptide is about one-tenth or less of that of a polypeptide containing a cytokine polypeptide generated by cleavage of the protease-cleavable linker. Some preferred fusion polypeptides are given by formulas (I) to (VI): [A]-[L1]-[H]-[L2]-[D](I); [D]-[L2]-[H]-[L1]-[A](II); [A]-[L1]-[D]-[L2]-[H](III); [H]-[L2]-[D]-[L1]-[A](IV); [H]-[L1]-[A]-[L2']-[D](V); [D]-[L1]-[A]-[L2']-[H](VI); [In the formula, A is a cytokine polypeptide, D is an inhibitory region, H is a half-life extension region, L1 is a protease-cleavable polypeptide linker, L2 is a polypeptide linker that can be selectively protease-cleaved, and L2' is a protease-cleavable polypeptide linker.] It is represented by one of the following. L1 and L2, or L1 and L2', may optionally have the same or different amino acid sequences and / or protease cleavage sites (if L2 is protease-cleavable). 【0008】 In some embodiments, the fusion proteins described herein are conditionally active variants of IL-12, IL-2, or IFN. In embodiments, the fusion protein may contain an IL-2 polypeptide. Fusion proteins containing an IL-2 polypeptide may contain or consist of the amino acid sequences of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646. The fusion proteins disclosed as SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646 are referred to herein as ACP289-ACP292, ACP296-ACP302, WW0301, ACP304-ACP306, ACP309-ACP313, and WW03 These are referred to as 53, ACP414, ACP336~ACP398, WW0472~WW0477, ACP406~ACP426, ACP439~ACP447, ACP451~ACP471, WW0729, WW0734~WW0792, ACP101, ACP293~ACP295, ACP316~ACP335, ACP427~ACP438, and ACP448~ACP450. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 272. The fusion protein may contain the amino acid sequence of SEQ ID NO: 286. The fusion protein may contain the amino acid sequence of SEQ ID NO: 362. The fusion protein may contain the amino acid sequence of SEQ ID NO: 336. The fusion protein may contain the amino acid sequence of SEQ ID NO: 348. The fusion protein may contain the amino acid sequence of SEQ ID NO: 363. The fusion protein may contain the amino acid sequence of SEQ ID NO: 580. 【0009】 In embodiments, the fusion protein may contain IL-12. A fusion protein containing an IL-12 polypeptide may contain or consist of one of the amino acid sequences of SEQ ID NOs. 368-371, 434-440, 453-519, or 523-538. The fusion proteins disclosed as SEQ ID NOs. 368-371, 434-440, 453-519, or 523-538 are referred to herein as ACP240-ACP245, ACP247, ACP285-ACP288, WW0641, WW0649-WW0652, WW0662-WW0725, WW0765-WW0772, and WW0796-WW0803. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 424. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 428. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 541. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 556. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 560. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 568. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 573. 【0010】 In embodiments, the fusion protein contains IFN. The fusion protein containing the IFN polypeptide may contain or consist of any one of the amino acid sequences of SEQ ID NOs. 421-430 and 539-578. The fusion proteins disclosed as SEQ ID NOs. 421-430 and 539-578 may be referred to herein as ACP200-ACP209, WW0644-WW0648, WW0781-WW0786, WW0815-WW0822, WW0831-WW0834, WW0737-WW0748 and WW0787-WW0790. 【0011】 In some embodiments, the fusion polypeptides disclosed herein may be covalently or noncovalently bound to a second polypeptide chain. For example, the fusion polypeptide may dimerize (i.e., form a dimer), or a portion of the fusion polypeptide may associate with another polypeptide, resulting in the formation of a functional binding site for, for example, a cytokine polypeptide or serum albumin. In some embodiments, the second polypeptide chain and the inhibitory portion on the fusion polypeptide are complementary and together form a functional binding site having specificity for the cytokine polypeptide contained in the fusion polypeptide. Exemplary functional binding sites that may be formed by an inhibitory portion of a fusion polypeptide and a complementary second polypeptide include antigen-binding sites of antibodies, such as the Fab fragment or a portion thereof of an antibody. For example, one chain of Fab that binds to a cytokine may be the inhibitory portion of the fusion polypeptide, e.g., VH-CH1, and the complementary VL-CL may be part of the second polypeptide. In such cases, the inhibitory portion of the fusion protein, namely VH-CH1, and the complementary second polypeptide containing VL-CL may associate to form a functional binding site that has specificity for cytokine polypeptides (e.g., IL-2, IL-12, IFN-alpha, IFN-beta) contained in the fusion protein, thereby weakening the cytokine polypeptide activity. 【0012】 In embodiments, the fusion protein containing the IL-2 cytokine polypeptide may be covalently or noncovalently bound to the second polypeptide chain. The second polypeptide chain may contain an antibody light chain VL-CL comprising or consisting of the amino acid sequence of SEQ ID NOs. 263, 264, or 333. Such a second polypeptide may be bound to a complementary VH-CH1 polypeptide contained in the fusion protein, for example, in SEQ ID NOs. 362, 363, 325, 286, 579, 581, or 582. The second polypeptide chains disclosed as SEQ ID NOs. 263, 264, and 333 may be referred to herein as WW0523(ACP381), WW0524(ACP382), or WW0556(ACP414). 【0013】In the embodiment, the fusion polypeptide may or may consist of the amino acid sequence of SEQ ID NOs: 362, 363, 325, 286, 579, 581, or 582, and the second polypeptide chain may or may consist of the amino acid sequence of SEQ ID NOs: 263, 264, or 333. The fusion polypeptides disclosed as SEQ ID NOs. 362, 363, 325, 286, 579, 581, or 582 may be designated as WW0520(ACP378), WW0521(ACP379), WW0548(ACP406), WW0621(ACP457), WW0729, WW0735, or WW0736, and the second polypeptide chains disclosed as SEQ ID NOs. 263, 264, and 333 may be designated herein as WW0523(ACP381), WW0524(ACP382), or WW0556(ACP414). For example, the fusion protein may contain or consist of the amino acid sequence of SEQ ID NOs. 362, and the second polypeptide chain may contain or consist of the amino acid sequence of SEQ ID NOs. 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264.For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 233. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 263. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 264. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 333. For example, the fusion protein may contain or be derived from SEQ ID NO: 582, and the second polypeptide chain may contain or be derived from SEQ ID NO: 263.For example, the fusion protein may contain or be derived from SEQ ID NO: 582, and the second polypeptide chain may contain or be derived from SEQ ID NO: 264. 【0014】 As described herein, target-direction is achieved by the action of an inhibitory polypeptide moiety that also binds to the desired target, or by a target-directing domain. A domain that recognizes a target antigen (e.g., tumor-specific antigen) attached to a preferred target may be ligated to the cytokine via a cleavable or non-cleavable linker. When ligated via a non-cleavable linker, the target-directing domain may further help retain the cytokine within the tumor and may be considered a retention domain. The target-directing domain does not necessarily have to be directly linked to the drug molecule; it may be directly linked to another element of the fusion protein. This is especially true when the target-directing domain is ligated via a cleavable linker. 【0015】 In one embodiment, a fusion polypeptide is provided comprising a cytokine polypeptide or a functional fragment, its mutain, its functional variant or subunit, and an inhibitory moiety, such as a steric inhibition domain. The inhibitory moiety is fused to the cytokine polypeptide directly or via a linker and can be separated from the cytokine polypeptide by cleavage (e.g., protease-mediated cleavage) of the fusion polypeptide at or near the fusion site, the linker, or within the inhibitory moiety. For example, if the cytokine polypeptide is fused to the inhibitory moiety via a linker containing a protease cleavage site, the cytokine polypeptide can be released from the inhibitory moiety via protease-mediated cleavage of the linker and bind to its receptor. The linker is designed to cleave at the desired cytokine action site, for example, in the tumor microenvironment, while avoiding off-target cytokine action and reducing the overall toxicity of cytokine therapy. 【0016】 The inhibitory portion may also function as a serum half-life extender. In some embodiments, the fusion polypeptide further comprises a separate serum half-life extender. In some embodiments, the fusion polypeptide further comprises a target-directed domain. In various embodiments, the serum half-life extender is a water-soluble polypeptide, such as optionally branched or multi-armed polyethylene glycol (PEG), full-length human serum albumin (HSA), or a fragment, Fc fragment, or nanobody that maintains binding affinity to FcRn, either directly to FcRn or to human serum albumin. 【0017】 In addition to serum half-life extending elements, the pharmaceutical compositions described herein preferably include at least one target-directed domain that binds to one or more target antigens, or to one or more regions on a single target antigen. It is intended herein that the polypeptide constructs of the present invention are cleaved at protease cleavage sites, for example, in the disease-specific microenvironment or the blood of the subject, and that the target-directed domain(s) bind to the target antigen on the target cell. At least one target antigen is involved in and / or associated with a disease, disorder, or pathological condition. Exemplary target antigens include those associated with proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases. 【0018】 In some embodiments, the target antigen is a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is located on tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, or fibrous tissue cells. 【0019】 Target antigens are, in some cases, expressed on the surface of diseased cells or tissues, such as tumor or cancer cells. Target antigens for tumors include, but are not limited to, fibroblast-activating protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling molecule 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, FAP, and CEA. The pharmaceutical compositions disclosed herein also include proteins comprising two antigen-binding domains that bind to two different target antigens known to be expressed on diseased cells or tissues. Exemplary pairs of antigen-binding domains include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3. 【0020】 In some embodiments, the target-directed polypeptide independently comprises an scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to a target antigen. In some embodiments, the target-directed polypeptide specifically binds to a cell surface molecule. In some embodiments, the target-directed polypeptide specifically binds to a tumor antigen. In some embodiments, the target-directed polypeptide specifically and independently binds to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide specifically and independently binds to two different antigens, at least one of which is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide acts as a retention domain and binds to a cytokine via a non-cleavable linker. 【0021】 As described herein, the cytokine-blocking moiety can bind to cytokines, thereby blocking the activation of homologous receptors of cytokines. 【0022】 This disclosure also relates to nucleic acids encoding conditionally active proteins as described herein, such as DNA, RNA, mRNA, and vectors and host cells containing such nucleic acids. 【0023】 This disclosure also relates to pharmaceutical compositions containing conditionally active proteins, nucleic acids encoding conditionally active proteins, and vectors and host cells containing such nucleic acids. Typically, the pharmaceutical composition contains one or more physiologically acceptable carriers and / or excipients. 【0024】 This disclosure also relates to a therapeutic method comprising administering an effective amount of a conditionally active protein, a nucleic acid encoding such a conditionally active protein, a vector or host cell containing such nucleic acid, and any of the above-mentioned pharmaceutical compositions to a subject requiring such treatment. Typically, the subject has or is at risk of developing a proliferative disorder, neoplastic disease, inflammatory disease, immunological disorder, autoimmune disease, infectious disease, viral disease, allergic reaction, parasitic reaction, graft-versus-host disease or host-versus-graft disease. 【0025】 This disclosure also relates to the use of conditionally active proteins, nucleic acids encoding conditionally active proteins, vectors or host cells containing such nucleic acids, and any of the above-mentioned pharmaceutical compositions for providing treatment in subjects in need thereof. Typically, subjects have or are at risk of developing proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases or host-versus-graft diseases. 【0026】 This disclosure also relates to the use of conditionally active proteins, nucleic acids encoding conditionally active proteins, vectors or host cells containing such nucleic acids for the manufacture of pharmaceuticals for treating diseases such as proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases or host-versus-graft diseases. [Brief explanation of the drawing] 【0027】 [Figure 1A] Figures 1A–1E are a series of graphs showing the activity of IL-2 fusion proteins in the HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and triangles represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of control IL-2. EC50 for each IL-12. The EC50 values ​​for each are shown in the table. Figures 1A–1E also show the results of protein cleavage assays for each IL-2 polypeptide. The test constructs shown include WW0475 (Figure 1A), WW0517 (Figure 1B), WW0548 / 556 (Figure 1C), WW0735 / 523 (Figure 1D), and WW0621 / 523 (Figure 1E). [Figure 1B] Figures 1A–1E are a series of graphs showing the activity of IL-2 fusion proteins in the HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and triangles represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of control IL-2. EC50 for each IL-12. The EC50 values ​​for each are shown in the table. Figures 1A–1E also show the results of protein cleavage assays for each IL-2 polypeptide. The test constructs shown include WW0475 (Figure 1A), WW0517 (Figure 1B), WW0548 / 556 (Figure 1C), WW0735 / 523 (Figure 1D), and WW0621 / 523 (Figure 1E). [Figure 1C]Figures 1A–1E are a series of graphs showing the activity of IL-2 fusion proteins in the HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and triangles represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of control IL-2. EC50 for each IL-12. The EC50 values ​​for each are shown in the table. Figures 1A–1E also show the results of protein cleavage assays for each IL-2 polypeptide. The test constructs shown include WW0475 (Figure 1A), WW0517 (Figure 1B), WW0548 / 556 (Figure 1C), WW0735 / 523 (Figure 1D), and WW0621 / 523 (Figure 1E). [Figure 1D] Figures 1A–1E are a series of graphs showing the activity of IL-2 fusion proteins in the HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and triangles represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of control IL-2. EC50 for each IL-12. The EC50 values ​​for each are shown in the table. Figures 1A–1E also show the results of protein cleavage assays for each IL-2 polypeptide. The test constructs shown include WW0475 (Figure 1A), WW0517 (Figure 1B), WW0548 / 556 (Figure 1C), WW0735 / 523 (Figure 1D), and WW0621 / 523 (Figure 1E). [Figure 1E] Figures 1A–1E are a series of graphs showing the activity of IL-2 fusion proteins in the HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and triangles represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of control IL-2. EC50 for each IL-12. The EC50 values ​​for each are shown in the table. Figures 1A–1E also show the results of protein cleavage assays for each IL-2 polypeptide. The test constructs shown include WW0475 (Figure 1A), WW0517 (Figure 1B), WW0548 / 556 (Figure 1C), WW0735 / 523 (Figure 1D), and WW0621 / 523 (Figure 1E). [Figure 1F]This graph shows the activity of the non-cleavable control WW0729 / 523. The results of the protein cleavage assay for the non-cleavable control are also shown. [Figure 2A] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2B] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2C]Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2D] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2E]Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2F] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2G]Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2H] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2I]Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2J] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2K]Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 2L] Figures 2A-2L are a series of graphs showing the activity of the fusion protein in the IL-2 luciferase reporter assay. Black squares represent the activity of the uncleaved IL-2 polypeptide (complete form), and white squares represent the activity of the cleaved IL-2 polypeptide (cleaved form). Circles represent the activity of the control (human IL-2). The EC50 values ​​for each are shown in the table (human IL-2). The test structures shown include WW0521 / WW0556 (Figure 2A), WW0521 / WW0524 (Figure 2B), WW0521 / WW0523 (Figure 2C), WW0520 / WW0524 (Figure 2D), WW0517 (Figure 2E), WW0516 (Figure 2F), WW0417 (Figure 2G), WW0317 (Figure 2H), WW0317 (Figure 2I), and WW0520 / WW0523 (Figure 2J), WW0621 / WW0523 (Figure 2K), and WW0048 (Figure 2L). [Figure 3A]Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3B] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3C] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3D]Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3E] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3F] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3G]Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3H] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3I] Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 3J]Figures 3A–3J are a series of graphs showing the activity of fusion proteins in the IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (complete form), triangles represent the activity of the cleaved polypeptide (cleaved form), and circles represent the activity of control human IL-2. The EC50 values ​​for each are shown in the table. The test constructs shown include WW0317 (Figure 3A), WW0516 (Figure 3B), WW0354 (Figure 3C), WW0517 (Figure 3D), WW0621 / 0523 (Figure 3E), WW0521 / 524 (Figure 3F), WW0520 / 0523 (Figure 3G), WW0729 / 523 (Figure 3H), and WW0735 / 523 (Figure 31), WW0520 / 524 (Figure 3J). [Figure 4A] Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4B] Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4C]Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4D] Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4E] Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4F] Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 4G]Figures 4A–4G are graphs showing the results of analyzing WW0475, WW0520 / 0523, WW0548 / 0524, WW0548 / 0556, WW0517, WW0621 / 0523, and WW0619 IL-2 fusion proteins in a syngeneic MC38 mouse tumor model. Each graph shows the mean tumor volume over time (mean + / - standard error) of mice treated with the different doses of each fusion protein shown. The data indicate that tumor volume decreases over time in a dose-dependent manner. [Figure 5A] Figures 5A–5C are a series of acronyms showing the activity of fusion proteins in an MC38 mouse syngeneic model, corresponding to the data shown in Figures 4A–4G. Each line in the graph represents the tumor volume over time in a single mouse. Figure 5A includes data corresponding to vehicle treatment and fusion proteins WW0517 and WW0520 / 523. [Figure 5B] Figures 5A–5C are a series of acronyms showing the activity of fusion proteins in MC38 mouse syngeneic models, corresponding to the data shown in Figures 4A–4G. Each line in the graph represents the tumor volume over time in a single mouse. Figure 5B includes data corresponding to treatment with fusion proteins WW0548 / 0524, WW0548 / 0556, and WW0475. [Figure 5C] Figures 5A–5C are a series of acronyms showing the activity of fusion proteins in an MC38 mouse syngeneic model, corresponding to the data shown in Figures 4A–4G. Each line in the graph represents the tumor volume over time in a single mouse. Figure 5C includes data corresponding to treatment with fusion proteins WW0619 and WW0621 / 0523. [Figure 6A] Figures 6A–6C are a series of arachnographs showing the effect of fusion proteins on body weight in an MC38 mouse syngeneic model, corresponding to the data shown in Figures 5A–5C. Each line in the graph represents the body weight of a single mouse over time. Figure 6A includes data corresponding to vehicle treatment and fusion proteins WW0517 and WW0520 / 523. [Figure 6B]Figures 6A–6C are a series of arachnographs showing the effect of fusion proteins on body weight in an MC38 mouse syngeneic model, corresponding to the data shown in Figures 5A–5C. Each line in the graph represents the body weight of a single mouse over time. Figure 6B includes data corresponding to treatment with fusion proteins WW0548 / 0524, WW0548 / 0556, and WW0475. [Figure 6C] Figures 6A–6C are a series of arachnographs showing the effect of fusion proteins on body weight in a syngeneic MC38 mouse model, corresponding to the data shown in Figures 5A–5C. Each line in the graph represents the body weight of a single mouse over time. Figure 6C includes data corresponding to treatment with fusion proteins WW0619 and WW0621 / 0523. [Figure 7A] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7B] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7C]Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7D] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7E] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7F]Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7G] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7H] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7I]Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7J] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7K] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7L]Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7M] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7N] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7O]Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7P] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show IL-12 / STAT4 activity compared between human p40 / mouse p35 IL-12 or human IL-12 fusion protein and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 7Q] Figures 7A–7Q are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figures 7A–7Q show the IL-12 / STAT4 activity of human p40 / mouse p35 IL-12 or human IL-12 fusion protein compared with chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 8A]Figures 8A–8D are a series of graphs showing the activity of fusion proteins in the IL12 T-Blast assay. They show the activation of IL-12 signaling compared between human p40 / mouse p35 IL12 or human IL12 fusion protein and control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of control chimeric IL-12 or recombinant human IL-12. The EC50 values ​​for each are shown in the table. [Figure 8B] Figures 8A–8D are a series of graphs showing the activity of fusion proteins in the IL12 T-Blast assay. They show the activation of IL-12 signaling compared between human p40 / mouse p35 IL12 or human IL12 fusion protein and control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of control chimeric IL-12 or recombinant human IL-12. The EC50 values ​​for each are shown in the table. [Figure 8C] Figures 8A–8D are a series of graphs showing the activity of fusion proteins in the IL12 T-Blast assay. They show the activation of IL-12 signaling compared between human p40 / mouse p35 IL12 or human IL12 fusion protein and control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of control chimeric IL-12 or recombinant human IL-12. The EC50 values ​​for each are shown in the table. [Figure 8D]Figures 8A–8D are a series of graphs showing the activity of fusion proteins in the IL12 T-Blast assay. They show the activation of IL-12 signaling compared between human p40 / mouse p35 IL12 or human IL12 fusion protein and control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (complete form), triangles represent the activity of the cleaved IL-12 polypeptide (cleaved form), and circles represent the activity of control chimeric IL-12 or recombinant human IL-12. The EC50 values ​​for each are shown in the table. [Figure 9A] Figures 9A-9C are a series of graphs showing the activity of fusion proteins in an IL-12 luciferase reporter assay. A-B show the activation of IL-12 signaling compared between the human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). C shows the activation of IL-12 signaling compared between the human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Black squares represent the activity of the uncleaved IL-12 polypeptide (complete form), and white squares represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 9B] Figures 9A-9C are a series of graphs showing the activity of fusion proteins in an IL-12 luciferase reporter assay. A-B show the activation of IL-12 signaling compared between the human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). C shows the activation of IL-12 signaling compared between the human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Black squares represent the activity of the uncleaved IL-12 polypeptide (complete form), and white squares represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 9C]Figures 9A-9C are a series of graphs showing the activity of fusion proteins in an IL-12 luciferase reporter assay. A-B show the activation of IL-12 signaling compared between the human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). C shows the activation of IL-12 signaling compared between the human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Black squares represent the activity of the uncleaved IL-12 polypeptide (complete form), and white squares represent the activity of the cleaved polypeptide (cleaved form). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. [Figure 10A] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10B] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10C] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10D] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10E]Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10F] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10G]Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10H] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10I]Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 10J] Figures 10A–10J are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. Figures 10A–10J show the activation of the IFN-α / β pathway compared between mouse INFα fusion protein and mouse INFα (control). Squares represent the activity of the uncleaved INFα polypeptide (complete form), triangles represent the activity of the cleaved INFα polypeptide (cleaved form), and circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. Figures 10A–10J also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 11A] Figures 11A-11B are graphs showing the results from the HEK-Blue IL-12 reporter assay performed on human p40 / mouse p35 IL-12 fusion proteins before and after protease cleavage. Constructs ACP35(A) and ACP34(B) were tested. Analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the activity of the IL-12 protein fusion protein. [Figure 11B]Figures 11A-11B are graphs showing the results from the HEK-Blue IL-12 reporter assay performed on human p40 / mouse p35 IL-12 fusion proteins before and after protease cleavage. Constructs ACP35(A) and ACP34(B) were tested. Analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results support the activity of the IL-12 protein fusion protein. [Figure 12A] Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 12B] Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 12C]Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 12D] Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 12E] Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 12F]Figures 12A–12F show a series of graphs illustrating the HEK-blue assay results for four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data indicate that cleaved IL-12 is more active than the complete fusion protein. The constructs tested were ACP06 (Figure 12A), ACP07 (Figure 12C), ACP08 (Figure 12B), ACP09 (Figure 12D), ACP10 (Figure 12E), and ACP11 (Figure 12F). [Figure 13] The results of the protein cleavage assay are shown. Both cleaved and uncleaved forms of the fusion protein ACP11 were flowed onto an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 14] This is a schematic diagram illustrating a non-restrictive example of an inducible cytokine protein, in which the construct is activated by protease cleavage of a linker bound between two subunits of the cytokine. [Figure 15A] Figures 15A–15D are graphs showing the results from HEK-Blue assays performed on human p40 / mouse p35 IL12 fusion proteins before and after protease cleavage. The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with or without HSA. [Figure 15B] Figures 15A–15D are graphs showing the results from HEK-Blue assays performed on human p40 / mouse p35 IL12 fusion proteins before and after protease cleavage. The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with or without HSA. [Figure 15C]Figures 15A–15D are graphs showing the results from HEK-Blue assays performed on human p40 / mouse p35 IL12 fusion proteins before and after protease cleavage. The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with or without HSA. [Figure 15D] Figures 15A–15D are graphs showing the results from HEK-Blue assays performed on human p40 / mouse p35 IL12 fusion proteins before and after protease cleavage. The results support the activity of the IL12 protein fusion protein. Each proliferation assay was performed with or without HSA. [Figure 16A] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16B] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16C] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16D] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16E] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16F] Figures 16A–16F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the WEHI279 cell survival assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17A] Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17B]Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17C] Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17D] Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17E] Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17F]Figures 17A–17F are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using the B16 reporter assay. Each assay was performed using either HSA-containing (+HSA) or HSA-free (-HSA) medium. Each fusion protein contained an anti-HSA binder, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 18A] Figures 18A-18B show the results of the protein cleavage assay described in Example 2. Two constructs, ACP31 (IFN-α fusion protein; A) and ACP55 (IFN-γ fusion protein; B), in both cleaved and uncleaved forms, were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 18B] Figures 18A-18B show the results of the protein cleavage assay described in Example 2. Two constructs, ACP31 (IFN-α fusion protein; A) and ACP55 (IFN-γ fusion protein; B), in both cleaved and uncleaved forms, were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 19A] Figures 19A–19B are a series of graphs (A and B) showing the activity of exemplary IFNγ fusion proteins before and after protease cleavage using the B16 reporter assay. Each assay was performed using culture medium containing HSA, and each fusion protein contained an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 19B] Figures 19A–19B are a series of graphs (A and B) showing the activity of exemplary IFNγ fusion proteins before and after protease cleavage using the B16 reporter assay. Each assay was performed using culture medium containing HSA, and each fusion protein contained an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 20A]Figures 20A–20B are a series of graphs (A and B) showing the activity of exemplary IFNα fusion proteins before and after cleavage using the B16 reporter assay. Each assay was performed using a medium containing HSA, and each fusion protein contained an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 20B] Figures 20A–20B are a series of graphs (A and B) showing the activity of exemplary IFNα fusion proteins before and after cleavage using the B16 reporter assay. Each assay was performed using a medium containing HSA, and each fusion protein contained an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 21A] Figures 21A–21D are a series of graphs showing the results of tumor growth studies using the MC38 cell line. A–C show the effects of intraperitoneal (IP) injection of IFNγ and IFNγ fusion protein using different dose levels and schedules (ug = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). D shows the effects of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 21B] Figures 21A–21D are a series of graphs showing the results of tumor growth studies using the MC38 cell line. A–C show the effects of intraperitoneal (IP) injection of IFNγ and IFNγ fusion protein using different dose levels and schedules (ug = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). D shows the effects of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 21C] Figures 21A–21D are a series of graphs showing the results of tumor growth studies using the MC38 cell line. A–C show the effects of intraperitoneal (IP) injection of IFNγ and IFNγ fusion protein using different dose levels and schedules (ug = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). D shows the effects of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 21D] Figures 21A–21D are a series of graphs showing the results of tumor growth studies using the MC38 cell line. A–C show the effects of intraperitoneal (IP) injection of IFNγ and IFNγ fusion protein using different dose levels and schedules (ug = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). D shows the effects of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 22A] Figures 22A–22B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP51(A) and ACP52(B)) cleaved by MMP9 protease using the B16 reporter assay, compared to the activity of the uncleaved fusion protein. Each fusion protein contains an anti-HSA binding agent and a tumor-targeting domain. [Figure 22B] Figures 22A–22B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP51(A) and ACP52(B)) cleaved by MMP9 protease using the B16 reporter assay, compared to the activity of the uncleaved fusion protein. Each fusion protein contains an anti-HSA binding agent and a tumor-targeting domain. [Figure 23A] Figures 23A–23B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease using the B16 reporter assay, compared to the activity of uncleaved fusion proteins. Each fusion protein contains IFNγ directly fused to albumin. [Figure 23B] Figures 23A–23B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease using the B16 reporter assay, compared to the activity of uncleaved fusion proteins. Each fusion protein contains IFNγ directly fused to albumin. [Figure 24A]Figures 24A-24D are graphs showing the results from the HEK-Blue IL-2 reporter assay performed on IL-2 fusion polypeptides and recombinant human IL-2 (Rec hIL-2). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24A shows the results for IL-2 constructs ACP132 and ACP133 with and without albumin. [Figure 24B] Figures 24A-24D are graphs showing the results from HEK-Blue IL-2 reporter assays performed on IL-2 fusion polypeptides and recombinant human IL-2 (Rec hIL-2). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24B shows the results for cleaved and uncleaved IL-2 construct ACP16. The results of protein cleavage assays for cleaved and uncleaved ACP16 are also shown. [Figure 24C] Figures 24A-24D are graphs showing the results from the HEK-Blue IL-2 reporter assay performed on IL-2 fusion polypeptides and recombinant human IL-2 (Rec hIL-2). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24C shows the results for the cleaved and uncleaved forms of the IL-2 construct ACP153. The results of the protein cleavage assay are also shown. [Figure 24D] Figures 24A-24D are graphs showing the results from the HEK-Blue IL-2 reporter assay performed on IL-2 fusion polypeptides and recombinant human IL-2 (Rec hIL-2). The analysis was performed based on the quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24D shows the results from the HEK-Blue IL-2 assay using wild-type cytokines, complete fusion proteins, and protease-cleaved fusion proteins. [Figure 25A]Figures 25A and 25B are two graphs showing the analysis of ACP16 (Figure 25A) and ACP124 (Figure 25B) in the HEKBlue IL-2 reporter assay in the presence of HSA. Circles represent the activity of the uncleaved polypeptide, and squares represent the activity of the cleaved polypeptide. [Figure 25B] Figures 25A and 25B are two graphs showing the analysis of ACP16 (Figure 25A) and ACP124 (Figure 25B) in the HEKBlue IL-2 reporter assay in the presence of HSA. Circles represent the activity of the uncleaved polypeptide, and squares represent the activity of the cleaved polypeptide. [Figure 25C] Figure 25C is a graph showing the results of the CTLL-2 proliferation assay. CTLL2 cells (ATCC) were seeded at a concentration of 500,000 cells / well suspended in culture medium with or without 40 mg / ml human serum albumin (HSA), and stimulated at 37°C and 5% CO2 for 72 hours with a dilution series of activatable hIL2. The activity of uncleaved and cleaved activatable ACP16 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell activity was evaluated using the CellTiter-Glo (Promega) luminescence-based cell viability assay. Circles represent complete fusion proteins, and squares represent protease-cleaved fusion proteins. [Figure 26A] Figures 26A–26C are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figure 26A shows the IL-12 / STAT4 activation compared between ACP11 (human p40 / mouse p35 IL12 fusion protein) and ACP04 (negative control). [Figure 26B] Figures 26A-26C are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figure 26B is a graph showing the analysis of ACP91 (chimeric IL-12 fusion protein). Squares represent the activity of the uncleaved ACP91 polypeptide, and triangles represent the activity of the cleaved polypeptide (ACP91 + MMP9). The EC50 values ​​for each are shown in the table. [Figure 26C]Figures 26A–26C are a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figure 26C is a graph showing the analysis of ACP136 (chimeric IL-12 fusion protein). Squares represent the activity of the uncleaved ACP136 polypeptide, and triangles represent the activity of the cleaved polypeptide (ACP136 + MMP9). The EC50 values ​​for each are shown in the inset table. [Figure 27A] Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27B] Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27C] Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27D] Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27E] Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27F]Figures 27A-27F are a series of graphs showing that the truncated mouse IFNα1 polypeptides ACP31 (Figure 27A), ACP125 (Figure 27B), and ACP126 (Figure 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 28A] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28B] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28C]Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28D] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28E] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28F]Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28G] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28H] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28I]Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28J] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28K] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28L]Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28M] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 28N] Figures 28A-28N are a series of graphs showing the activity of ACP56 (Figure 28A), ACP57 (Figure 28B), ACP58 (Figure 28C), ACP59 (Figure 28D), ACP60 (Figure 28E), ACP61+HSA (Figure 28F), ACP30+HSA (Figure 28G), ACP73 (Figure 28H), ACP70+HSA (Figure 28I), ACP71 (Figure 28J), ACP72 (Figure 28K), ACP73 (Figure 28L), ACP74 (Figure 28M), and ACP75 (Figure 28N) in the B16 IFNγ reporter assay. The activity of each fusion was tested both when cleaved (square) and when uncleaved (circle). [Figure 29A]Figures 29A and 29B are two graphs showing the results of analyzing ACP31 (mouse IFNα1 fusion protein) and ACP11 (human p40 / mouse p35 IL12 fusion protein) in a tumor xenograft model. Figure 29A shows the tumor volume over time in mice treated with 33 μg of ACP31 (circles), 110 μg of ACP31 (triangles), 330 μg of ACP31 (diamonds), and controls of 1 μg of mouse wild-type IFNα1 (dashed line, squares) and 10 μg of mIFNα1 (dashed line, small circles). The vehicle alone is represented by a large white circle. The data show that tumor volume decreases over time in a dose-dependent manner in mice treated with ACP31. [Figure 29B] Figures 29A and 29B are two graphs showing the results of analyzing ACP31 (mouse IFNα1 fusion protein) and ACP11 (human p40 / mouse p35 IL12 fusion protein) in a tumor xenograft model. Figure 29B shows the tumor volume over time in mice treated with 17.5 μg of ACP11 (square), 175 μg of ACP31 (triangle), 525 μg of ACP31 (circle), and as controls, 2 μg of ACP04 (dashed line, triangle) and 10 μg of ACP04 (dashed line, diamond). The vehicle alone is represented by a large white circle. The data show that tumor volume decreases over time in a dose-dependent manner in both mice treated with ACP11 and ACP04 (human p40 / mouse p35 IL12 fusion protein). [Figure 30A] Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 30B]Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 30C] Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 30D] Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 30E] Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 30F] Figures 30A–30F are a series of line graphs showing the time course of tumor volume in mouse xenograft tumor models of mice treated with vehicle alone (Figure 30A), 2 μg of ACP04 (Figure 30B), 10 μg of ACP04 (Figure 30C), 17.5 μg of ACP11 (Figure 30D), 175 μg of ACP11 (Figure 30E), and 525 μg of ACP11 (Figure 30F), respectively. Each line represents a single mouse. [Figure 31A]Figures 31A-31C are three graphs showing the results of analyzing ACP16 and ACP124 in a tumor xenograft model. Figure 31A shows the tumor volume over time in mice treated with 4.4 μg of ACP16 (square), 17 μg of ACP16 (triangle), 70 μg of ACP16 (inverted triangle), 232 μg of ACP16 (black circle), and control drugs 12 μg of wild-type IL-2 (dashed line, triangle) and 36 μg of wild-type IL-2 (dashed line, diamond). The vehicle alone is represented by a large white circle. The data shows that the tumor volume decreases over time in a dose-dependent manner in mice treated with the higher concentration of ACP16. [Figure 31B] Figures 31A-31C are three graphs showing the results of analyzing ACP16 and ACP124 in a tumor xenograft model. Figure 31B shows the tumor volume over time in mice treated with 17 μg of ACP124 (square), 70 μg of ACP124 (triangle), 230 μg of ACP124 (inverted triangle), and 700 μg of ACP124. The vehicle alone is represented by a large white circle. [Figure 31C] Figures 31A-31C are three graphs showing the results of analyzing ACP16 and ACP124 in a tumor xenograft model. Figure 31C shows the tumor volume over time in mice treated with 17 μg of ACP16 (triangle), 70 μg of ACP16 (circle), 232 μg of ACP16 (black circle), and the control drugs 17 μg of ACP124 (dashed line, triangle), 70 μg of ACP124 (dashed line, diamond), and 230 μg of ACP124 (dashed line, diamond). The vehicle alone is represented by a black inverted triangle. The data show that the tumor volume decreased over time in a dose-dependent manner in mice treated with ACP16 rather than ACP124. [Figure 32A] Figures 32A–32B are a series of line graphs showing the activity of fusion proteins in the MC38 mouse xenograft model, corresponding to the data shown in Figure 31. Each line in the graph represents a single mouse. [Figure 32B] Figures 32A–32B are a series of line graphs showing the activity of fusion proteins in the MC38 mouse xenograft model, corresponding to the data shown in Figure 31. Each line in the graph represents a single mouse. [Figure 33] Figure 33 is a graph showing the time course of tumor volume in a mouse xenograft model illustrating tumor growth in control mice (white circles) and AP16-treated mice (squares). [Figure 34A] Figures 34A–34D are a series of survival graphs showing the time course of survival in mice treated with cleavable fusion proteins. Figure 34A shows data for mice treated with vehicle alone (gray line), 17 μg of ACP16 (black line), and 17 μg of ACP124 (dashed line). [Figure 34B] Figures 34A–34D are a series of survival graphs showing the time course of survival in mice treated with cleavable fusion proteins. Figure 34B shows data for mice treated with vehicle alone (gray line), 70 μg of ACP16 (black line), and 70 μg of ACP124 (dashed line). [Figure 34C] Figures 34A–34D are a series of survival graphs showing the time course of survival in mice treated with cleavable fusion proteins. Figure 34C shows data for mice treated with vehicle alone (gray line), 232 μg of ACP16 (black line), and 230 μg of ACP124 (dashed line). [Figure 34D] Figures 34A–34D are a series of survival graphs showing the time course of survival in mice treated with cleavable fusion proteins. Figure 34D shows data for mice treated with vehicle alone (gray line), 232 μg of ACP16 (black line), and 700 μg of ACP124 (dashed line). [Figure 35] A series of line graphs showing the activity of fusion proteins in an MC38 mouse xenograft model. All mouse groups received a total of four doses, except for APC132, which received up to three doses at which lethal toxicity was detected after two doses per week. Shown are vehicle alone (top), ACP16 at 17, 55, 70, and 230 μg (top row), ACP132 at 9, 28, 36, and 119 μg (middle row), and ACP21 at 13, 42, 54, and 177 μg (bottom row). Each line in the graph represents an individual animal. [Figure 36A]Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36B] Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36C]Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36D] Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36E]Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36F] Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36G]Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 36H] Figures 36A–36H are a series of graphs showing the activity of fusion proteins in the HEK-Blue IFN-α / β reporter assay. Figures 36A–36H show the activation of the IFN-α / β pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control (human IFNα). The EC50 values ​​for each are shown in the table. The results support the idea that the IFNα fusion protein is active and inducible. Figures 36A–36H also show the results of the protein cleavage assay for each INFα fusion protein. Both cleaved and uncleaved forms of each INFα fusion protein were run on an SDS-PAGE gel. As seen on the gel, the cleavage was complete. [Figure 37A]Figures 37A–37D are a series of graphs showing the activity of the fusion protein in the HEK-Blue IFN-α / β reporter assay. Figures 37A–37B show the activation of the IFN-α / β pathway compared between the mouse INFβ fusion protein and the control (mouse IFNβ). Figures 37C–37D show the activation of the IFN-α / β pathway compared between the human INFβ fusion protein and the control (human IFNβ). Squares represent the activity of the uncleaved INFβ polypeptide (complete form), triangles represent the activity of the cleaved INFβ polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results support the finding that the IFNβ fusion protein is active and inducible. [Figure 37B] Figures 37A–37D are a series of graphs showing the activity of the fusion protein in the HEK-Blue IFN-α / β reporter assay. Figures 37A–37B show the activation of the IFN-α / β pathway compared between the mouse INFβ fusion protein and the control (mouse IFNβ). Figures 37C–37D show the activation of the IFN-α / β pathway compared between the human INFβ fusion protein and the control (human IFNβ). Squares represent the activity of the uncleaved INFβ polypeptide (complete form), triangles represent the activity of the cleaved INFβ polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results support the finding that the IFNβ fusion protein is active and inducible. [Figure 37C] Figures 37A–37D are a series of graphs showing the activity of the fusion protein in the HEK-Blue IFN-α / β reporter assay. Figures 37A–37B show the activation of the IFN-α / β pathway compared between the mouse INFβ fusion protein and the control (mouse IFNβ). Figures 37C–37D show the activation of the IFN-α / β pathway compared between the human INFβ fusion protein and the control (human IFNβ). Squares represent the activity of the uncleaved INFβ polypeptide (complete form), triangles represent the activity of the cleaved INFβ polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results support the finding that the IFNβ fusion protein is active and inducible. [Figure 37D]Figures 37A–37D are a series of graphs showing the activity of the fusion protein in the HEK-Blue IFN-α / β reporter assay. Figures 37A–37B show the activation of the IFN-α / β pathway compared between the mouse INFβ fusion protein and the control (mouse IFNβ). Figures 37C–37D show the activation of the IFN-α / β pathway compared between the human INFβ fusion protein and the control (human IFNβ). Squares represent the activity of the uncleaved INFβ polypeptide (complete form), triangles represent the activity of the cleaved INFβ polypeptide (cleaved form), and circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results support the finding that the IFNβ fusion protein is active and inducible. [Figure 38A] Figures 38A-38C are a series of graphs showing the activity of the fusion protein in a human PBMC assay. A-C show the activation of the IFNα pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control human IFNα. The EC50 values ​​for each are shown in the table. The analysis was performed based on the quantification of CXCL-10 (IP-10). The results support the finding that the IFNα fusion protein is active and inducible. [Figure 38B] Figures 38A-38C are a series of graphs showing the activity of the fusion protein in a human PBMC assay. A-C show the activation of the IFNα pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control human IFNα. The EC50 values ​​for each are shown in the table. The analysis was performed based on the quantification of CXCL-10 (IP-10). The results support the finding that the IFNα fusion protein is active and inducible. [Figure 38C]Figures 38A-38C are a series of graphs showing the activity of the fusion protein in a human PBMC assay. A-C show the activation of the IFNα pathway compared between the human IFNα fusion protein and the control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (complete form), triangles represent the activity of the cleaved IFNα polypeptide (cleaved form), and circles represent the activity of the control human IFNα. The EC50 values ​​for each are shown in the table. The analysis was performed based on the quantification of CXCL-10 (IP-10). The results support the finding that the IFNα fusion protein is active and inducible. [Figure 39A] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39A shows the average tumor volume over time in mice treated with 369 μg of WW0610 (square), 553 μg of WW0610 (downward triangle), 830 μg of WW0610 (upward triangle), and 1,245 μg of WW0610 (circle). The vehicle alone is represented by a black circle. [Figure 39B] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39B shows the mean tumor volume over time in mice treated with 1,231 μg of WW0815 (square), 1,845 μg of WW0815 (downward triangle), 2,770 μg of WW0815 (upward triangle), and 4,154 μg of WW0815 (circle). The vehicle alone is represented by a black circle. [Figure 39C] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39C shows the average tumor volume over time in mice treated with 4.6 μg of WW0644 (square), 9.3 μg of WW0644 (downward triangle), 19 μg of WW0644 (upward triangle), and 37 μg of WW0644 (circle). The vehicle alone is represented by a black circle. [Figure 39D]Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39D shows the mean tumor volume over time in mice treated with 31 μg of WW0816 (square), 62 μg of WW0816 (downward triangle), 123 μg of WW0816 (upward triangle), and 247 μg of WW0816 (circle). The vehicle alone is represented by a black circle. [Figure 39E] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39E shows the mean tumor volume over time in mice treated with 110 μg of WW0609 (square), 830 μg of WW0609 (downward triangle), and 1,320 μg of WW0609 (upward triangle). [Figure 39F] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39F shows the mean tumor volume over time in mice treated with 110 μg of WW0610 (square), 830 μg of WW0610 (downward triangle), and 1,320 μg of WW0610 (upward triangle). [Figure 39G] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39G shows the average tumor volume over time in mice treated with 0.3 μg of WW0643 (square), 1.5 μg of WW0643 (downward triangle), 7.5 μg of WW0643 (upward triangle), and 37.5 μg of WW0643 (diamond). The vehicle alone is represented by a black circle. [Figure 40A] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40B] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40C]Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40D] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40E] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40F] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 40G] Figures 40A–40G show a series of acronyms illustrating the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A–39G. Each line in the graph represents the tumor volume over time in a single mouse. [Figure 41A] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41B] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41C] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41D] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41E] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41F] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 41G] Figures 41A–41G show the average body weight percentage over time for mice treated with the INF fusion protein shown in Figures 39A–39G. [Figure 42A] Figures 42A–42E show the results of the B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after cleavage. The relevant wild-type IFN was tested as a control. [Figure 42B] Figures 42A–42E show the results of the B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after cleavage. The relevant wild-type IFN was tested as a control. [Figure 42C] Figures 42A–42E show the results of the B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after cleavage. The relevant wild-type IFN was tested as a control. [Figure 42D] Figures 42A–42E show the results of the B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after cleavage. The relevant wild-type IFN was tested as a control. [Figure 42E] Figures 42A–42E show the results of the B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after cleavage. The relevant wild-type IFN was tested as a control. [Figure 43] The connectivity data for ACP16, ACP10, and ACP11 is shown. [Figure 44A] Figures 44A to 44D show the activity of the cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244, and ACP247. [Figure 44B] Figures 44A to 44D show the activity of the cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244, and ACP247. [Figure 44C]Figures 44A to 44D show the activity of the cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244, and ACP247. [Figure 44D] Figures 44A to 44D show the activity of the cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244, and ACP247. [Figure 45] A series of arachnographs showing tumor volume over time during treatment with vehicle, IL-12, ACP11, or ACP10 are shown. [Figure 46A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 46B] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 46C] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 46D] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 47A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 47B]Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 47C] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 47D] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 48A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 48B] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49B] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49C] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49D] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49E] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49F] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49G] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49H] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 49I] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 50A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 50B]Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 51A] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 51B] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 51C] Figures 46A–46D, 47A–47D, 48A, A8B, A9A–49I, 50A, 50B, and 51A–51C show data (tumor volume and / or body weight) for mice treated with cytokine fusion protein constructs. [Figure 52A] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52B] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52C] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52D] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52E] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52F] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52G] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52H]Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52I] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52J] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52K] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52L] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52M] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 52N] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 53A] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 53B] Figures 52A-52N, 53A, and 53B show the activity of cytokine fusion protein constructs. [Figure 54A] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54B] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54C] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54D] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54E]Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54F] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54G] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54H] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54I] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54J] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54K] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54L] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54M] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 54N] Figures 54A–54N show the results of a proliferation assay comparing cleaved protein, uncleaved protein, and IL2 as a control. [Figure 55A] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55B]Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55C] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55D] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55E] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55F] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55G] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55H] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55I] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55J] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55K]Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55L] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55M] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 55N] Figures 55A–55N show the results of a HekBlue IL2 reporter assay comparing the activity of constructs with and without protease cleavage, with IL-2 included as a control. [Figure 56] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57A] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57B] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57C] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57D] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 58] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59A] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59B] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59C] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59D] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59E] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59F] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59G] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59H] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59I] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59J] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59K] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59L] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59M] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59N] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59O] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59P]Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Q] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59R] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59S] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59T] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59U] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59V] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59W] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Z-1] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Y] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Z-2] Figures 56, 57A-57D, 58, and 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 60A] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60B]Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60C] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60D] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60E] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60F] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 60G] Figures 60A–60G show a series of arachnographs illustrating the effect of INF fusion protein on body weight in MC38 mouse xenograft models, corresponding to the data shown in Figures 41A–41G. Each line in the graph represents the body weight of a single mouse over time. [Figure 61A]Figures 61A and 61B are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. A shows the activation of the IFN-α / β pathway for construct WW0609 in the presence and absence of albumin. B shows the activation of the IFN-α / β pathway for construct WW0643 in the presence and absence of albumin. Squares represent the activity of the uncleaved INFα polypeptide (complete form), and triangles represent the activity of the cleaved INFα polypeptide (cleaved form). Circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. [Figure 61B] Figures 61A and 61B are a series of graphs showing the activity of fusion proteins in the B16-Blue IFN-α / β reporter assay. A shows the activation of the IFN-α / β pathway for construct WW0609 in the presence and absence of albumin. B shows the activation of the IFN-α / β pathway for construct WW0643 in the presence and absence of albumin. Squares represent the activity of the uncleaved INFα polypeptide (complete form), and triangles represent the activity of the cleaved INFα polypeptide (cleaved form). Circles represent the activity of the control (mouse IFNα). The EC50 values ​​for each are shown in the table. [Modes for carrying out the invention] 【0028】 This specification discloses methods and compositions for designing and using constructs containing inducible cytokines. Cytokines are potent immune agonists, and for this reason, they are considered promising therapeutic agents for oncology. However, cytokines have proven to have a very narrow therapeutic window. Cytokines have a short serum half-life and are considered to be very potent. As a result, therapeutic administration of cytokines has produced undesirable systemic effects and toxicity. These have been exacerbated by the need to administer large amounts of cytokine to achieve the desired level of cytokine at the intended site of action (e.g., tumor). Unfortunately, due to the biology of cytokines, as well as the inability to effectively direct and control their activity, cytokines have not delivered the much-anticipated clinical benefits in the treatment of tumors. 【0029】 This specification discloses a fusion protein that overcomes the challenges of toxicity and short half-life that have significantly limited the clinical use of cytokines in oncology. The fusion protein contains a cytokine polypeptide having receptor agonist activity. However, in the context of the fusion protein, the cytokine receptor agonist activity is weakened and the circulating half-life is extended. The fusion protein includes a protease cleavage site that is associated with a desired cytokine site of action (e.g., tumor) and is typically cleaved by a protease that is abundant or selectively present at the desired site of action. Thus, the fusion protein is preferentially (or selectively) and efficiently cleaved at the desired site of action, substantially limiting the cytokine activity to the desired site of action, e.g., the tumor microenvironment. Protease cleavage at the desired site of action, e.g., the tumor microenvironment, releases from the fusion protein a form of cytokine that is far more active as a cytokine receptor agonist than the fusion protein (typically at least about 100 times more active than the fusion protein). The cytokines released upon cleavage of fusion proteins typically have short half-lives, often substantially the same as those of naturally occurring cytokines, resulting in more localized cytokine activity within the tumor microenvironment. Despite the extended half-life of the fusion protein, toxicity is dramatically reduced or eliminated due to the attenuation of the circulating fusion protein and the direction of active cytokines into the tumor microenvironment. The fusion proteins described herein enable, for the first time, the administration of effective therapeutic doses of cytokines for treating tumors, where cytokine activity is substantially limited to the tumor microenvironment, and unwanted systemic effects and toxicity of cytokines are dramatically reduced and eliminated. 【0030】 The fusion proteins disclosed herein typically comprise a cytokine polypeptide [A], an inhibitory moiety [D], an optional half-life extension moiety [H], and a protease-cleavable polypeptide linker. The cytokine polypeptide, the inhibitory moiety, and the optional half-life extension element (if present) are functionally linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has a weakened cytokine receptor activating effect, for example, the cytokine receptor activating effect of the fusion polypeptide is about one-tenth or less of that of a polypeptide containing the cytokine polypeptide generated by cleavage of the protease-cleavable linker. Some preferred fusion polypeptides are given by formulas (I) to (VI): [A]-[L1]-[H]-[L2]-[D](I); [D]-[L2]-[H]-[L1]-[A](II); [A]-[L1]-[D]-[L2]-[H](III); [H]-[L2]-[D]-[L1]-[A](IV); [H]-[L1]-[A]-[L2']-[D](V); [D]-[L1]-[A]-[L2']-[H](VI); [In the formula, [A] is a cytokine polypeptide, [D] is an inhibitory region, [H] is a half-life extension region, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that can be selectively protease-cleaved, and [L2'] is a protease-cleavable polypeptide linker.] It is represented by one of the following: [L1] and [L2], or [L1] and [L2'] may optionally have the same or different amino acid sequences and / or protease cleavage sites (if L2 is protease-cleavable). 【0031】 This disclosure further relates to pharmaceutical compositions containing inducible fusion proteins and additional therapeutic agents, as well as nucleic acids encoding polypeptides, and recombinant expression vectors and host cells (sells) for producing such fusion proteins. Methods for using the fusion proteins of this disclosure in the treatment of diseases, conditions, and disorders are also provided herein. 【0032】 Unless otherwise specified, all terms of the art, symbolism, and other scientific and technical knowledge used herein are intended to have the meanings commonly understood by those skilled in the art. In some cases, terms having commonly understood meanings are defined herein for clarity and / or for ease of reference, and the inclusion of such definitions herein should not necessarily be interpreted as representing a difference from the commonly understood meanings in the art. The techniques and procedures described or referred to herein are generally well understood and are commonly employed by those skilled in the art using conventional methodologies, such as the molecular cloning methodology described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is widely used. Procedures involving the use of commercially available kits and reagents, where necessary, should generally be carried out according to the manufacturer's specified protocols and conditions unless otherwise noted. 【0033】 "Cytokines" is a well-known technical term referring to any of the classes of immunomodulatory proteins (e.g., interleukins or interferons) secreted by immune system cells and acting as regulators of the immune system. Cytokine polypeptides that may be used in the fusion proteins disclosed herein include transforming growth factors, e.g., TGF-α and TGF-β (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3); interferons, e.g., interferon-α, interferon-β, interferon-γ, interferon-kappa and interferon-omega; and interleukins, e.g., IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11. Examples include, but are not limited to, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25; tumor necrosis factors, e.g., tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., CXC motif chemokines 10 (CXCL10), CCL19, CCL20, CCL21), as well as granulocyte-macrophage colony-stimulating factors (GM-CS), and fragments of polypeptides that activate syngeneic receptors for cytokines (i.e., the functional fragments mentioned above). "Chemokine" is a technical term referring to any family of small cytokines that have the ability to induce targeted chemotaxis in nearby responding cells. 【0034】 Cytokines are well known to have short serum half-lives, often only a few minutes or hours. Even cytokines in modified amino acid sequences intended to extend their serum half-life while retaining receptor agonist activity typically have short serum half-lives. As used herein, “short half-life cytokine” refers to cytokines having a relatively short half-life circulating in the serum of the subject, e.g., less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 60 minutes, less than 90 minutes, less than 120 minutes, less than 240 minutes, or less than 480 minutes. As used herein, short half-life cytokines include cytokines that have not been sequence-modified to result in polypeptides having modified amino acid sequences intended to produce a longer-than-normal half-life in the subject's body and to extend their serum half-life while retaining receptor agonist activity. The latter does not imply the addition of heterologous protein domains, e.g., genuine half-life-extending elements, e.g., serum albumin. 【0035】 A "sortase" is a peptidyltransferase that modifies a protein by recognizing and cleaving a carboxyl terminal sorting signal embedded in or bound to the end of a target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 442) (where X is any standard amino acid) on the target protein between the Thr and Gly residues, which involves the transient binding of the Thr residue to the Cys residue at the active site on the enzyme to form an enzyme-thioacyl intermediate. To complete the peptide transfer and produce a peptide-monomer conjugate, a biomolecule with an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, replacing sortase A and linking the two molecules together. 【0036】 As used herein, the term "steric blocker" refers to a polypeptide or polypeptide moiety that is covalently bound to a cytokine polypeptide, either directly or indirectly through another moiety such as a linker, e.g., in the form of a chimeric polypeptide (fusion protein), or is otherwise not covalently bound to the cytokine polypeptide. A steric blocker can be non-covalently bound to a cytokine polypeptide, e.g., by electrostatic, hydrophobic, ionic, or hydrogen bonding. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity and relative size to the cytokine moiety. A steric blocker can also effect inhibition by employing a large protein binding partner. An example of this is an antibody that binds to serum albumin, where the antibody itself may or may not be large enough to activate or block binding on its own, while employing albumin enables sufficient steric hindrance. 【0037】 As used and described herein, a "half-life extension element" is a part of a chimeric polypeptide that increases the serum half-life and improves pK by altering parameters such as its size (e.g., exceeding the renal filtration cut-off), shape, hydrodynamic radius, charge, or absorption, biodistribution, metabolism, and excretion. 【0038】 As used herein, the terms "activatable", "activate", "induce", and "inducible" mean the ability of a protein that is part of a fusion protein, i.e., a cytokine, to bind to its receptor and exert an effect upon cleavage of an additional element from the fusion protein. 【0039】 As used herein, a "plasmid" or "viral vector" is an agent that transports nucleic acids of the present disclosure into cells without degradation and contains a promoter that results in the expression of cellular nucleic acid molecules and / or polypeptides in the cells to which it is delivered. 【0040】 As used herein, the terms "peptide", "polypeptide" or "protein" are used broadly to mean two or more amino acids linked by peptide bonds. Proteins, peptides and polypeptides are also used interchangeably herein to refer to amino acid sequences. It should be recognized that the term polypeptide is not used herein to imply a particular size or number of amino acids constituting the molecule, and that the peptides of the present invention may contain fewer or more than a few amino acid residues. 【0041】 As used throughout, "subject" can be a vertebrate, and more specifically, can be a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and neonatal subjects are intended to be included whether male or female. 【0042】 As used herein, "patient" or "subject" can be used interchangeably and can refer to a subject having a disease or disorder (e.g., cancer). The terms patient or subject include human and veterinary subjects. 【0043】 As used herein, the terms “treatment,” “to treat,” or “to treat” mean a method of reducing the effects of a disease or condition, or the symptoms of a disease or condition. Accordingly, in the methods of this disclosure, treatment may mean a reduction of the severity of an existing disease or condition, or the symptoms of a disease or condition, by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially all of it. For example, a method of treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Accordingly, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the original level or control level. It is understood that treatment does not necessarily mean the cure or complete elimination of the disease, condition, or symptoms of the disease or condition. 【0044】 As used herein, the terms “prevent,” “prevent,” and “prevent” a disease or disorder mean an action that occurs before or at approximately the time when a subject begins to exhibit one or more symptoms of a disease or disorder, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, which suppresses or delays the onset or exacerbation of one or more symptoms of the disease or disorder. 【0045】 As used herein, references to “reduce,” “amount decrease,” or “suppress” include changes of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or greater, compared to a preferred control level. Such terms may, but are not necessarily, include the complete elimination of a function or characteristic, such as agonist action. 【0046】 A "weakened cytokine receptor agonist" is a cytokine receptor agonist that has reduced receptor agonist activity compared to the naturally occurring agonists of the cytokine receptor. A weakened cytokine agonist may have agonist activity that is at most about 1 / 10, at most about 1 / 50, at most about 1 / 100, at most about 1 / 250, at most about 1 / 500, at most about 1 / 1000, or less, compared to the naturally occurring agonist of the receptor. When a fusion protein containing a cytokine polypeptide described herein is described as "weakened" or having "weakened activity," it means that the fusion protein is a weakened cytokine receptor agonist. 【0047】 A "complete fusion protein" is a fusion protein in which no domains have been removed, for example, by protease cleavage. Domains can be removed by protease cleavage or other enzymatic action, but this does not happen when the fusion protein is "complete". 【0048】 As used herein, “part” refers to a portion of a molecule that has a distinct function, and that function may be exerted by that part in the context of another molecule. A part may be a chemical entity having a specific function, or a part of a biomolecule having a specific function. For example, a “blocking portion” in a fusion protein is a portion of the fusion protein that can block some or all of the activity of the fusion polypeptide. This may be a protein domain, such as serum albumin. Blockage can be achieved by a steric inhibitor or a specific inhibitor. A steric inhibitor blocks by size and location rather than by specific binding, and serum albumin is an example. A specific inhibitor blocks by specific interaction with the portion to be blocked. A specific inhibitor must be formulated to fit a specific cytokine or active domain, while a steric inhibitor can be used independently of the onboard drug, as long as it is sufficiently large. If desired, a blocking portion incorporated into a fusion protein as described herein may associate with another polypeptide to create a specific binding domain. For example, when using a cytokine-binding fragment of an antibody as the inhibitory site, the fusion polypeptide may contain a cytokine-specific scFv as the inhibitory site, or the fusion polypeptide may contain half of a Fab, such as VH-CH1 or VL-CL, as the inhibitory site, which may associate with the complementary VL-CL or VH-CH1 chain, respectively, to form a Fab fragment that specifically binds to cytokines. As further described herein, the inhibitory site may inhibit the activity of the fusion protein directly or when the inhibitory site is associated with another polypeptide, for example, when an anti-HSA scFv is bound to HSA, or when a VH-CH1 polypeptide associates with a complementary VL-CL polypeptide and subsequently binds to a cytokine polypeptide. 【0049】 In general, the therapeutic use of cytokines is severely limited by their systemic toxicity. For example, TNF was initially discovered for its ability to induce hemorrhagic necrosis in some tumors and for its in vitro cytotoxic effects on various oncological lineages, but it was later proven to have a strong pro-inflammatory effect that can endanger the human body under conditions of overproduction. Because systemic toxicity is a fundamental challenge to the use of pharmacologically active cytokines in humans, novel derivatives and therapeutic strategies are currently being evaluated with the aim of mitigating the toxic effects while maintaining the therapeutic efficacy of this type of biological effector. 【0050】 IL-2 plays both stimulative and regulatory roles in the immune system and, along with other members of the common gamma chain (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R), which consists of either a trimer composed of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains, or a dimer βγIL-2R(1,3). Both IL-2R variants can transmit signals through binding to IL-2. However, the trimer αβγIL-2R has approximately 10 to 100 times higher affinity for IL-2 compared to the dimer βγIL-2R(3), suggesting that CD25 contributes to the high affinity binding of IL-2 to its receptor but is not essential for signal transduction. Trimeric IL-2R is found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ T regulatory cells (Tregs) that are sensitive to IL-2, both in vitro and in vivo. Conversely, antigen-sensitized (memory) CD8+, CD44 high-memory phenotype (MP) CD8+, and natural killer (NK) cells have high levels of dimeric βγIL-2R, and these cells also respond strongly to IL-2 both in vitro and in vivo. 【0051】 The expression of high-affinity IL-2R is crucial for T cells to respond to low concentrations of IL-2 that are transiently available in vivo. IL-2Rα expression is absent on native and memory T cells but is induced after antigen activation. IL-2Rβ is constitutively expressed on NK, NKT, and memory CD8+ T cells, but is also induced on native T cells after antigen activation. γc is regulated much more slowly and is constitutively expressed on all lymphoid cells. Once high-affinity IL-2R is induced by an antigen, IL-2R signaling partially upregulates IL-2Rα expression through Stat5-dependent regulation of IL2ra transcription (Kim et al., 2001). This process represents a mechanism that maintains high-affinity IL-2R expression and sustains IL-2 signaling as long as a source of IL-2 remains. 【0052】 IL-2 is captured by IL-2Rα via a broad hydrophobic binding surface surrounded by polar margins, resulting in a relatively weak interaction (Kd 10-8M) with a fast binding-dissociation kinetics. However, the IL-2Rα-IL-2 binary complex imparts a very small conformational change to IL-2 that facilitates association with IL-2Rβ through separate polar interactions between IL-2 and IL-2Rβ. As shown in Ciardelli's data, the pseudohigh affinity (i.e., Kd approximately 300 pM) of the IL2 / α / β trimer complex clearly indicates that the trimer complex is more stable than either IL2 bound only to the α chain (Kd=10 nM) or IL2 bound only to the β chain (Kd=450 nM). In any case, the IL2 / α / β trimer then recruits the γ chain into a quaternary complex capable of signal transduction, facilitated by a large complex binding site on the IL2-binding β chain to the γ chain. 【0053】 In other words, the ternary IL-2Rα-IL-2Rβ-IL-2 complex then recruits γc through weak interactions with IL-2 and stronger interactions with IL-2Rβ to produce a stable quaternary high-affinity IL-2R (Kd 10-11M, i.e., 10pM). The formation of the high-affinity quaternary IL-2-IL-2R complex leads to signaling by tyrosine kinases Jak1 and Jak3, which associate with IL-2Rβ and γc, respectively (Nelson and Willerford, 1998). The quaternary IL-2-IL-2R complex is rapidly internalized, and IL-2, IL-2Rβ, and γc are rapidly degraded, while IL-2Rα is recycled on the cell surface (Hemar et al., 1995; Yu and Malek, 2001). Therefore, functional actions that require sustained IL-2R signaling necessitate a continuous supply of IL-2 to engage with IL-2Rα and form further IL-2-IL-2R signaling complexes. 【0054】 Interleukin-15 (IL-15), another member of the 4-alpha-helix bundle family of cytokines, also emerged as an immunomodulator for cancer treatment. IL-15 is initially captured by IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages. IL-15 exerts broad-spectrum effects, inducing differentiation and proliferation of T, B, and natural killer (NK) cells through signaling via IL-15 / IL-2-R-β (CD122) and the common gamma chain (CD132). It also interacts with CD8 + Enhances the cytolytic effect of T cells and prolongs antigen sensitization CD8 +IL-15 induces CD44 memory T cells. IL-15 stimulates B cell differentiation and immunoglobulin synthesis and induces dendritic cell maturation. It does not stimulate immunosuppressive T regulatory cells (Tregs). Therefore, selective enhancement of IL-15 activity in the tumor microenvironment may enhance innate and specific immunity to fight tumors (Waldmann et al., 2012). IL-15 was initially identified for its ability to stimulate T cell proliferation in a manner similar to IL-2, via its common receptor component (IL-2R / 15Rβ-γc), and through signaling by JAK1 / JAK3 and STAT3 / STAT5. Like IL-2, IL-15 has been shown to stimulate the proliferation of activated CD4-CD8-, CD4+CD8+, CD4+, and CD8+ T cells, as well as to facilitate the induction of cytotoxic T lymphocytes and the generation, proliferation, and activation of NK cells (Waldmann et al., 1999). However, unlike IL-2, which is required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and to retain these cells in the periphery, IL-15 has little effect on Tregs (Berger et al., 2009). This is important because FOXP3-expressing CD4+CD25+ Tregs suppress effector T cells, thereby inhibiting immune responses, including those targeting tumors. While IL-2 also plays a crucial role in initiating activation-induced cell death (AICD), a process that leads to the removal of autoreactive T cells, IL-15 is an anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000). IL-15 co-delivered with HIV peptide vaccines has been shown to overcome CD4+ T cell deficiency by promoting the persistence of antigen-specific CD8+ T cells and inhibiting TRAIL-mediated apoptosis (Oh et al., 2008). Furthermore, IL-15 promotes the long-term maintenance of CD8+CD44hi memory T cells (Kanegane et al., 1996). 【0055】 The importance of IL-15 and IL-15Rα for T and NK cell development is evident in IL-15Rα - / - and IL-15 - / - This is further emphasized by the mouse phenotype. Knockout mice show a reduction in the total number of CD8+ T cells, and several subsets of memory phenotype CD8+ T cells, NK cells, NK / T cells, and intercellular lymphocytes of the intestinal epithelium are deficient, indicating that IL-5 provides essential positive homeostatic functions for these subsets of cells (Lodolce et al., 1996; Kennedy et al., 1998). The phenotypic similarities between these two strains of knockout mice suggest the importance of IL-15Rα in maintaining physiologically important IL-15 signaling. 【0056】 IL-15 is presented in trans to the IL-15Rβγc complex on the surface of T cells and natural killer (NK) cells via the IL-15 receptor alpha chain (Han et al., 2011). The IL-15Rα chain acts as a chaperone protein, stabilizing and increasing IL-15 activity (Desbois et al., 2016). Exogenous IL-15 has been shown to have limited effects on cancer patients due to its dependence on IL-15Rα, which is often downregulated in cancer patients. Therefore, the fusion protein RLI, composed of the sushi+ domain of IL15Ra linked to IL-15 via a linker, has been proposed as an alternative to IL-15 therapy (Bessard et al., 2009). Administration of soluble IL-15 / IL-15Rα complexes was found to significantly enhance the half-life and bioavailability of IL-15 in serum in vivo (Stoklasek et al., 2010). 【0057】 In addition to its effects on T and NK cells, IL-15 also has several effects on other components of the immune system. IL-15 protects neutrophils from apoptosis, modulates phagocytosis, and stimulates the secretion of IL-8 and IL-1R antagonists. It functions by activating JAK2, p38, and ERK1 / 2 MAPK, Syk kinase, and NF-κB transcription factors (Pelletier et al., 2002). In mast cells, IL-15 can act as a growth factor and an inhibitor of apoptosis. In these cells, IL-15 activates the JAK2 / STAT5 pathway without requiring γc binding (Tagaya et al., 1996). IL-15 also induces B lymphocyte proliferation and differentiation and increases immunoglobulin secretion (Armitage et al., 1995). It also prevents Fas-mediated apoptosis and allows for the induction of an antibody response partially independent of CD4 assistance (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages, and dendritic cells effectively transcribe and translate IL-15. They also respond to IL-15 stimulation. Macrophages respond by increasing phagocytosis, inducing IL-8, IL-12, and MCP-1 expression, and secreting IL-6, IL-8, and TNFα (Budagian et al., 2006). Dendritic cells incubated with IL-15 show maturation with increased CD83, CD86, CD40, and MHC class II expression, and are even resistant to apoptosis and show enhanced interferon-γ secretion (Anguille et al., 2009). 【0058】 IL-15 has also been shown to have effects on non-blood cells, including monocytes, adipocytes, endothelial cells, and nerve cells. IL-15 has anabolic effects on muscle and may support muscle cell differentiation (Quinn et al., 1995). It can stimulate muscle cells and muscle fibers to accumulate contractile proteins and slow muscle wasting in rats with cancer-associated cachexia (Figeus et al., 2004). IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce microglial growth and survival (Hanisch et al., 1997). 【0059】 Interleukin-7 (IL-7), also belonging to the IL-2 / IL-15 family, is a well-characterized, multifaceted cytokine expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, and keratinocytes, as well as by dendritic cells after activation (Alpdogan et al., 2005). Initially described as a growth and differentiation factor for precursor B lymphocytes, later studies have shown that IL-7 is critically involved in T lymphocyte development and differentiation. Interleukin-7 signaling is essential for the establishment of optimal CD8 T cell function, homeostasis, and memory (Schluns et al., 2000), and it has been proposed that it is required by most T cell subsets and that its expression is important for regulating T cell number. 【0060】 IL-7 is IL-7Rα and γ cIL-7Rα binds to dimeric receptors, forming a ternary complex that plays a fundamental role in extracellular matrix rearrangement, T and B cell development, and homeostasis (Mazzucchelli and Durum, 2007). IL-7Rα also cross-reacts to form a ternary complex with thymic stromal lymphocyte necrosis factor (TSLP) and its receptor (TSLPR), activating the TSLP pathway and leading to T and dendritic cell proliferation in humans, as well as B cell development in mice (Leonard, 2002). Therefore, tight regulation of the signaling cascade activated by the complex is crucial for normal cellular function. Mutations in the IL-7Rα external domain cause attenuation of the IL-7 pathway, inhibiting T and B cell development and resulting in patients with a form of severe combined immunodeficiency (SCID) (Giliani et al., 2005; Puel et al., 1998). 【0061】 IL-7 has a potential role in enhancing immune rearrangement in cancer patients after cytotoxic chemotherapy. IL-7 therapy can enhance immune rearrangement and even improve limited thymic function by facilitating peripheral proliferation, even for a small number of recently emerged thymic cells. Therefore, IL-7 therapy may be able to repair the immune system of patients depleted by cytotoxic chemotherapy (Capitini et al., 2010). 【0062】 Interleukin-12 (IL-12) is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40), and these subunits are covalently linked to generate a so-called bioactive heterodimeric (p70) molecule (Lieschke et al., 1997, Jana et al., 2014). In addition to forming heterodimers (IL-12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and a homodimer (p402). It is known in the art that synthesis of the heterodimer as a single chain by a linker connecting p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays a crucial role in the initial inflammatory response to infection and in the generation of Th1 cells, which favorably act on cell-mediated immunity. It has been found that overproduction of IL-12 can be dangerous to the host as it is involved in the development of multiple autoimmune inflammatory diseases (e.g., MS, arthritis, type 1 diabetes). 【0063】 The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T cells and natural killer cells (Trinchieri et al., 2003). The IL-12Rβ1 chain binds to the IL-12p40 subunit, while IL-12p35 associated with IL-12Rβ2 confers intracellular signaling ability (Benson et al., 2011). Signaling by IL-12R induces phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), which phosphorylate and activate signal transducer and activator of transcription (STAT) 1, STAT3, STAT4, and STAT5. The specific cellular actions of IL-12 are mainly due to the activation of STAT4. IL-12 induces natural killer and T cells to produce cytokines, particularly interferon (IFN) γ, which mediate many of the pro-inflammatory actions of IL-12, including the differentiation of CD4+ T cells into the Th1 phenotype (Montepaone et al., 2014). 【0064】 Regulatory T cells actively suppress the activation of the immune system, preventing pathological autoreactivity and the resulting autoimmune diseases. Developing drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune diseases has been a subject of extensive research, and was generally unsuccessful until the development of the present invention, which can selectively deliver activated interleukins to the site of inflammation. Regulatory T cells (Tregs) are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Tregs are central to immune system homeostasis and play a major role in maintaining resistance to autoantigens and regulating immune responses to foreign antigens. Several autoimmune and inflammatory diseases, including type 1 diabetes (T1D), systemic lupus erythema (SLE), and graft-versus-host disease (GVHD), have been shown to be associated with deficiencies in Treg cell count or Treg function. 【0065】 Consequently, there is considerable interest in developing therapies that enhance the number and / or function of Treg cells. One therapeutic approach being studied for autoimmune diseases is the transplantation of autologous, ex vivo grown Treg cells (Tang, Q., et al, 2003, Cold Spring Harb. Perspect. Med., 3:1-15). While this approach has shown promise in treating animal models of the disease and in several early-stage human trials, it requires personalized therapy with the patient's own T cells, is invasive, and is technically complex. Another approach is treatment with low-dose interleukin-2 (IL-2). Treg cells are characterized by the high constitutive expression of the high-affinity IL-2 receptor IL2Rαβγ, which is composed of the subunits IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), and Treg cell growth has been shown to be IL-2 dependent (Malek, TR, et al., 2010, Immunity, 33:153-65). 【0066】 Conversely, immunostimulation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) is approved for the treatment of certain cancers. High-dose IL-2 is used for the treatment of patients with metastatic melanoma and metastatic renal cell carcinoma, with long-term effects on overall survival. 【0067】 Clinical trials of low-dose IL-2 therapy in patients with chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) demonstrated elevated Treg levels and indicated clinical efficacy. New clinical trials have been initiated to investigate the efficacy of IL-2 in several other autoimmune and inflammatory diseases. The rationale for using so-called low-dose IL-2 was to utilize the high IL-2 affinity of the trimer IL-2 receptor constitutively expressed on Tregs, while keeping other T cells that do not express the high-affinity receptor inactive. Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, CA) is a recombinant form of IL-2 used in these trials, but it has been associated with high toxicity. Aldesleukin is approved at high doses for the treatment of metastatic melanoma and metastatic renal cancer, but its side effects are very severe, so its use is only recommended in hospital settings where intensive care is available (web address: www.proleukin.com / assets / pdf / proleukin.pdf). 【0068】 Clinical trials of IL-2 in autoimmune diseases have employed lower doses of IL-2 to target Treg cells, because Treg cells respond to lower concentrations of IL-2 compared to many other immune cell types due to their expression of IL2R-alpha (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower doses have presented safety and tolerability issues, and the treatments used have involved daily subcutaneous injections, either for extended periods or in intermittent 5-day treatment cycles. Therefore, there is a need for autoimmune disease therapies that enhance Treg cell number and function, target Treg cells more specifically than IL-2, are safer and more tolerable, and can be administered at lower frequencies. 【0069】 One proposed approach to improve the therapeutic index of IL-2-based therapies for autoimmune diseases is to use variants of IL-2 that are more selective to Treg cells than to other immune cells. The IL-2 receptor is expressed on a diverse range of immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad mode of expression may be responsible for its multifaceted effects on the immune system and its high systemic toxicity. In particular, activated T effector cells express IL2Rαβγ as well as lung epithelial cells. However, activating T effector cells directly contradicts the goal of modulating and controlling the immune response to weaken it, and activating lung epithelial cells leads to known dose-limiting side effects of IL-2, such as pulmonary edema. In fact, the major side effect of high-dose IL-2 immunotherapy is vascular leakage syndrome (VLS), which leads to intravascular fluid accumulation in organs such as the lungs and liver, followed by pulmonary edema and hepatocyte damage. There is no treatment for VLS other than discontinuation of IL-2. Low-dose IL-2 treatment plans have been tested in patients to avoid VLS, but this came at the cost of suboptimal treatment outcomes. 【0070】 According to the literature, VLS is thought to be caused by the release of pro-inflammatory cytokines from IL-2 activated NK cells. However, there is some evidence that pulmonary edema is due to the direct binding of IL-2 to pulmonary endothelial cells that express low to moderate levels of functional αβγIL-2R. And, by blocking binding to CD25 using an anti-CD25 monoclonal antibody (mAb) in CD25-deficient host mice, or by using a CD122-specific IL-2 / anti-IL-2 mAb (IL-2 / mAb) complex, pulmonary edema associated with the interaction between IL-2 and pulmonary endothelial cells was eliminated, and thus VLS was prevented. 【0071】 Treatment with interleukin cytokines other than IL-2 has become increasingly limited. IL-15 exerts similar immune cell stimulating effects to IL-2, but does not have the same inhibitory effects, making it a promising immunotherapy candidate. Clinical trials of recombinant human IL-15 for the treatment of metastatic melanoma or renal cell carcinoma demonstrated significant changes in immune cell distribution, proliferation, and activation, suggesting potential antitumor activity (Conlon et al., 2014). IL-15 is currently in clinical trials to treat various forms of cancer. However, IL-15 therapy is known to be associated with undesirable adverse effects, such as certain progressive leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver injury. (Mishra A., et al., Cancer Cell,2012,22(5):645-55, Alpdogan O. et al.,Blood,2005,105(2):866-73, Conlon KC et al.,J Clin Oncol,2015,33(1):74-82). 【0072】 IL-7 promotes lymphocyte development in the thymus and maintains the persistence of naive and memory T cell homeostasis in the periphery. Furthermore, it is important for organogenesis of lymph nodes (LNs) and for the maintenance of activated T cells recruited into secondary lymphoid organs (SLOs) (Gao et al., 2015). In clinical trials of IL-7, patients receiving IL-7 showed increases in both CD4+ and CD8+ T cells without a significant increase in regulatory T cell counts, as tracked by FoxP3 expression (Sportes et al., 2008). In clinical trials reported in 2006, 2008, and 2010, patients with various cancers, such as metastatic melanoma or sarcoma, received subcutaneous injections of different doses of IL-7. Toxicity was minimal, other than transient fever and mild erythema. Circulating levels of CD4+ and CD8+ T cells significantly increased, and the number of Tregs decreased. TCR repertoire diversity increased after IL-7 therapy. However, the antitumor activity of IL-7 has not been adequately evaluated (Gao et al., 2015). The results suggest that IL-7 therapy may enhance and expand the immune response. 【0073】 IL-12 is a multifaceted cytokine whose action creates interconnections between innate and adaptive immunity. IL-12 was first described as a factor secreted from PMA-induced EBV-transformed B cell lines. Based on its action, IL-12 is named cytotoxic lymphocyte maturation factor and natural killer cell stimulator. Due to its ability to bridge the gap between innate and adaptive immunity and its potent stimulation of IFNγ production—a cytokine that harmonizes natural anti-cancer defense mechanisms—IL-12 appeared to be an ideal candidate for tumor immunotherapy in humans. However, serious side effects associated with systemic administration of IL-12 in clinical trials, and the very narrow therapeutic index of this cytokine, have significantly dampened enthusiasm for its use in cancer patients (Lasek et al., 2014). A tumor-targeted approach to IL-12 therapy, which may mitigate some of the conventional problems associated with IL-12 therapy, is currently in clinical trials for cancer. 【0074】 The direct use of IL-2 as an agonist that binds to IL-2R and therapeutically modulates the immune response is problematic due to well-supported therapeutic risks in the literature, such as its short serum half-life and high toxicity. These risks have also limited the development and use of therapeutic agents of other cytokines. There is a need for new forms of cytokines that mitigate these risks. This specification discloses compositions and methods comprising IL-2 and IL-15, as well as other cytokines, functional fragments and mutaines of cytokines, variants and subunits of cytokines, and conditionally active cytokines, which have been devised to address these risks and provide necessary immunomodulatory therapeutic agents. 【0075】 The present invention is designed to address the shortcomings of direct IL-2 therapy and therapies using other cytokines, using, for example, cytokine-inhibiting moieties, such as steric-inhibiting polypeptides, serum half-life-extending polypeptides, target-directed polypeptides, linking polypeptides, such as protease-cleavable linkers, and combinations thereof. Cytokines, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFN including IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factor (e.g., TNF-alpha, lymphotoxin), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), chemokines (CXC-motif chemokines 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage-colony-stimulating factor (GM-CS), are highly potent when administered to patients. As used herein, “chemokine” means a family of small cytokines that have the ability to induce targeted chemotaxis in nearby responsive cells. Cytokines can provide potent therapies but are associated with undesirable effects that are difficult to control clinically and have limited the clinical use of cytokines. This disclosure relates to novel forms of cytokines that can be used for patients with reduced or eliminated undesirable effects. In particular, this disclosure relates to pharmaceutical compositions comprising chimeric polypeptides (fusion proteins), nucleic acids encoding fusion proteins, and the above-mentioned pharmaceutical formulations comprising cytokines or active fragments of cytokines or muteins having reduced cytokine receptor activating activity compared to the corresponding cytokines. Nevertheless, under selected conditions or in selected biological environments, chimeric polypeptides often activate their homologous receptors at the same or higher titers as the corresponding naturally occurring cytokines. As described herein, this is typically achieved by using cytokine-inhibiting moieties that block or inhibit the receptor-activating function of cytokines, their active fragments or muteins under normal conditions but not under selected conditions, such as those present at the desired cytokine site of action (e.g., inflammatory sites or tumors). 【0076】 Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be prepared using any preferred method. For example, nucleic acids encoding chimeric polypeptides can be prepared using recombinant DNA technology, synthetic chemistry, or a combination of these technologies and can be expressed in a preferred expression system, such as CHO cells. Chimeric polypeptides can similarly be prepared by the expression of preferred nucleic acids, such as synthetic or semi-synthetic chemical technologies. In some embodiments, the inhibitory moiety can be conjugated to the cytokine polypeptide by sortase-mediated conjugation. A "sortase" is a peptidyltransferase that modifies a protein by recognizing and cleaving a carboxyl-end sorting signal embedded in or bound to the end of a target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 442) (where X is any standard amino acid) on a target protein between the Thr and Gly residues, which involves the transient binding of the Thr residue to the active site Cys residue on the enzyme to form an enzyme-thioacyl intermediate. To complete the peptide transfer and create a peptide-monomer conjugate, a biomolecule with an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, replacing sortase A and linking the two molecules together. 【0077】 To form cytokine-blocking moiety fusion proteins, the cytokine polypeptide is first tagged with a polyglycine sequence at its N-terminus or with an LPXTG motif (SEQ ID NO: 442) at its C-terminus. The blocking moiety or other element is then bound to the respective peptide, which acts as a receptor site for the tagged polypeptide. For N-terminal conjugation to a domain containing the bound LPXTG motif (SEQ ID NO: 442) receptor peptide, the polypeptide is tagged with a sequence of N-terminal polyglycines. For C-terminal conjugation to a domain containing the polyglycine peptide, the polypeptide is tagged with an LPXTG (SEQ ID NO: 442) sortase recognition sequence at its C-terminus. The sortase recognizes the polyglycine and LPXTG (SEQ ID NO: 442) sequences, forming a peptide bond between the polymer-peptide and the tagged polypeptide. The sortase reaction cleaves a glycine residue as an intermediate and occurs at room temperature. 【0078】 Various mechanisms may be employed to eliminate or reduce the inhibition caused by the blocking site. For example, a pharmaceutical composition may include a cytokine moiety and a blocking site, such as a steric blocking site, such that a protease-cleavable linker containing a protease cleavage site is located between or within the cytokine blocking site and the cytokine blocking site. When the protease cleavage site is cleaved, the blocking site may dissociate from the cytokine, and the cytokine may then activate the cytokine receptor. The cytokine moiety may also be blocked by a specific blocking site, such as an antibody that binds to an epitope found on the cytokine in question. 【0079】 Any suitable linker may be used. For example, the linker may be glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (Sequence ID 443) or (Gly3Ser) nThis may include (SEQ ID NO: 444), where n is 1, 2, 3, 4, or 5. Typically, the sortase recognition motif includes the peptide sequence LPXTG (SEQ ID NO: 442), where X is any amino acid. In some embodiments, there is a covalent linkage between a reactive lysine residue bound to the C-terminus of the cytokine polypeptide and a reactive aspartate residue bound to the N-terminus of the inhibitor or other domain. In other embodiments, there is a covalent linkage between a reactive aspartate residue bound to the N-terminus of the cytokine polypeptide and a reactive lysine residue bound to the C-terminus of the inhibitor or other domain. 【0080】 Therefore, as described in detail herein, the cytokine-inhibiting moiety used may be a steric inhibitor. As used herein, “steric inhibitor” refers to a polypeptide or polypeptide moiety that may be covalently bound to the cytokine polypeptide, either directly or indirectly via other parts such as a linker, for example, in the form of a chimeric polypeptide (fusion protein), but otherwise is not covalently bound to the cytokine polypeptide. A steric inhibitor may be bound to the cytokine polypeptide non-covalently, for example, by electrostatic, hydrophobic, ionic, or hydrogen bonds. Typically, a steric inhibitor inhibits or blocks the activity of the cytokine moiety due to its proximity to and relative size to the cytokine moiety. Steric inhibition of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric inhibitor, for example, by enzymatically cleaving a fusion protein containing the steric inhibitor and cytokine peptide at a site between the steric inhibitor and the cytokine polypeptide. 【0081】 As described further herein, the inhibitory function may be due to the combination of additional functional components such as target-directed domains, serum half-life extension elements, and protease-cleavable linked polypeptides, or due to their presence in the pharmaceutical composition. For example, serum half-life extension polypeptides may also be steric inhibitors. 【0082】 To provide a concise disclosure of the overall perspective of the present invention, embodiments of the invention will be described in detail using the cytokine IL-2 as an exemplary cytokine. However, the present invention and this disclosure are not limited to IL-2. It will be apparent to those skilled in the art that this disclosure, including the methods, polypeptides and nucleic acids of this disclosure, fully describes and enables the use of other cytokines, fragments, manifolds, cytokine subunits and mutains, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha, lymphotoxin, TGF-beta-1, TGF-beta-2, TGF-beta-3, GM-CSF, CXCL10, CCL19, CCL20, CCL21, and any of the functional fragments or mutains described above. Preferred cytokines for use in the fusion proteins disclosed herein are IL-2, IL-12, IFN-alpha, IFN-beta, IFN-gamma, any of the mutains described above, functional manifolds and functional fragments or subunits. For example, the cytokine IL-12 can be a p35 subunit, a p40 subunit, or a heterodimer. 【0083】 Various factors ensure the delivery and activation of IL-2 at the desired IL-2 active site, and severely limit systemic exposure to interleukins by inhibitory and / or targeted strategies preferentially associated with serum half-life extension strategies. In these serum half-life extension strategies, the inhibited form of interleukin circulates for an extended period (preferably 1-2 weeks or more), while the activated form has the typical serum half-life of interleukin. 【0084】 Compared to forms with extended serum half-lives, intravenously administered IL-2 has a serum half-life of only about 10 minutes due to its distribution into a large extracellular space of approximately 15 L in an average-sized adult. Subsequently, IL-2 is metabolized by the kidneys with a half-life of approximately 2.5 hours (Smith, K. "Interleukin 2 immunotherapy." Therapeutic Immunology 240 (2001)). Other measurements indicate that IL-2 has a very short plasma half-life of 85 minutes after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of the present invention, the half-life extension element is linked to the interleukin via a linker, which is cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full action of the interleukin at the desired site, and further separate it from the uncleaved half-life extension. In such embodiments, the fully active and free interleukin will have very different pharmacokinetic (pK) properties—half-lives of several hours instead of several weeks. In addition, exposure to the active cytokine is limited to the desired cytokine site of action (e.g., inflammation site or tumor), reducing systemic exposure to the active cytokine and associated toxicity and side effects. 【0085】 Other cytokines conceivable in the present invention have pharmacological properties similar to IL-2 (e.g., IL-15 as reported by Blood 2011 117:4787-4795;doi:doi.org / 10.1182 / blood-2010-10-311456), and therefore the design of the present invention addresses the drawbacks of using these agents directly and provides chimeric polypeptides that may have an extended half-life and / or be directed to a desired active site (e.g., a site of inflammation or tumor). 【0086】 If desired, IL-2 may be manipulated to bind to the IL-2R complex as a whole, or specifically to one of the three IL-2R subunits, with an affinity different from that corresponding to wild-type IL-2, for example, by selectively activating Treg or Teff. For example, an IL-2 polypeptide that is said to have a higher affinity for the trimer form of the IL-2 receptor than for the dimeric beta / gamma form of the IL-2 receptor compared to wild-type IL-2 may have an amino acid sequence containing one of the following sets of mutations relating to Sequence ID No. 1 (a mature IL-2 protein containing amino acids 21-153 of human IL-2 with Uniprot accession number P60568-1): (a) K64R, V69A and Q74P; (b) V69A, Q74P and T101A; (c) V69A, Q74P and I128T; (d) N30D, V69A, Q74P and F103S; (e) K 49E, V69A, A73V and K76E; (f)V69A, Q74P, T101A and T133N; (g)N30S, V69A, Q74P and I128A; (h)V69A, Q74P, N88D and S99P; (i)N30S, V69A, Q74P and I128T; (j)K9T, Q11R, K3 5R, V69A and Q74P; (k)A1T, M46L, K49R, E61D, V69A and H79R; (l)K48E, E68D, N71T, N90H, F103S and I114V; (m)S4P, T10A, Q11R, V69A, Q74P, N88D and T133A; (n)E15K, N30S Y31H, K35R, K48E, V69A, Q74P and I92T; (o)N30S, E68D, V69A, N71A, Q74P, S75P, K76R and N90H; (p)N30S, Y31C, T37A, V69A, A73V, Q74P, H79R and I128T; (q)N26D, N29S, N3 0S, K54R, E67G, V69A, Q74P and I92T; (r)K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P and I92T; and (s)N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D and I89V.This method can also be applied to prepare mutains of other cytokines, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs including IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factor (e.g., TNF-alpha, lymphotoxin), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), and granulocyte-macrophage-colony-stimulating factor (GM-CS). For example, mutains with a desired binding affinity to a synonym receptor can be prepared. 【0087】 As stated above, any of the mutant IL-2 polypeptides disclosed herein may include the described sequences; they may also be limited to the described sequences and other sequences identical to SEQ ID NO: 1. Furthermore, any of the mutant IL-2 polypeptides disclosed herein may optionally include a substitution of the cysteine ​​residue at position 125 of SEQ ID NO: 1 with another residue (e.g., serine), and / or an optional deletion of the alanine residue at position 1. 【0088】 Another approach to improving the therapeutic index of IL-2-based therapy is to optimize the molecular pharmacokinetics to maximize Treg cell activation. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro requires exposure to effective concentrations of IL-2 for at least 5–6 hours (Cantrell, DA, et al., 1984, Science, 224:1312–1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes for intravenous administration and 3.3 hours for subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5–10). Due to its short half-life, maintaining circulating IL-2 above the level required to stimulate T cell proliferation for the required duration necessitates either high doses or frequent administrations that result in peak IL-2 levels significantly above the EC50 for Treg cells. These high IL-2 peak levels may activate the IL2Rβγ receptor and have other unintended or adverse effects, such as the VLS described above. IL-2 analogs, or multifunctional proteins with IL-2 bound to their domain, which have a longer circulating half-life than IL-2 and enable binding to the FcRn receptor, can achieve target drug concentrations over a specific period at lower doses and lower peak levels than IL-2. Therefore, such IL-2 analogs require lower doses or lower frequency administration compared to IL-2 to effectively stimulate Treg cells. Lower frequency subcutaneous administration of IL-2 drugs also makes them more tolerable for patients. Therapeutic agents with these characteristics clinically lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy. Alternatively, IL-2 or IL-2 mutaine (hereinafter referred to as "IL-2") may be used. *“) can be selectively directed to the intended site of action (e.g., an inflammatory site or a tumor). This targeting can be achieved by one of several strategies, including the addition of a domain to the administered agent that blocks the truncated IL-2 (or mutein), or by a targeting domain, or by a combination of both. 【0089】 In some embodiments, IL-2 is adapted to have a higher or lower affinity depending on the desired target. * A partial agonist can be made, for example, IL-2 * can be engineered to bind to one of the receptor subunits with enhanced affinity but not to the others. These types of partial agonists, unlike full agonists or full antagonists, provide the ability to modulate signaling properties to a magnitude that elicits desired functional characteristics without reaching the threshold of undesirable characteristics. Considering the differential activity of partial agonists, it may also be possible to engineer the repertoire of IL-2 variants to exhibit unique signaling activities with a finer degree of activation ranging from almost full to partial agonistic action to full antagonistic action. 【0090】 In some embodiments, IL-2 * has a modified affinity for IL-2Rα. In some embodiments, IL-2 * has a higher affinity for IL-2Rα than wild-type IL-2. In other embodiments, IL-2 * has a changed affinity for IL-2Rβ. In one embodiment, IL-2 * has an enhanced binding affinity for IL-2Rβ, e.g., the N-terminus of IL-2Rβ, thereby eliminating the functional requirement for IL-2Rα. In another embodiment, an IL-2 is generated that has an increased binding affinity for IL-2Rβ but exhibits a reduced binding to IL-2Rγ, thereby preventing normal functioning of IL-2Rβγ heterodimerization and signaling. * 【0091】 The inhibitory portion, as described in more detail below, can also be used to favor binding to or activation of one or more receptors. In one embodiment, an inhibitory portion is added that blocks the binding or activation of IL-2Rβγ but leaves no change in the binding or activation of IL-2Rα. In another embodiment, an inhibitory portion is added that weakens the binding or activation of IL-2Rα. In yet another embodiment, an inhibitory portion is added that inhibits the binding to or activation of all three receptors. This inhibition may be released by removing the inhibitory portion in a specific environment, for example, by proteolytic cleavage of the linker linking one or more inhibitory portions to the cytokine. 【0092】 Similar techniques may be applied to improve other cytokines, particularly for use as immunostimulants for the treatment of cancer. For example, in this embodiment, the pharmacokinetics and / or pharmacodynamics of cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFN-alpha, IFN-beta and IFN-gamma, TNF-alpha, lymphotoxin, TGF-beta-1, TGF-beta-2, TGF-beta-3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21) may be modified to maximize the activation of effector cells (e.g., effector T cells, NK cells) and / or cytotoxic immune response-promoting cells (e.g., to induce dendritic cell maturation) at a desired site of action, preferably not systemically, such as within a tumor. 【0093】 Thus, pharmaceutical compositions comprising at least one cytokine polypeptide, e.g., interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs including IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxins), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), chemokines (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage-colony-stimulating factor (GM-CS), or any of the above functional fragments or mutains, are provided herein. The polypeptide typically also includes at least one linker amino acid sequence, which in some embodiments can be cleaved by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence including HSSKLQ (SEQ ID NO: 25), GPLVGRG (SEQ ID NO: 445), IPVSLRSG (SEQ ID NO: 446), VPLSLYSG (SEQ ID NO: 447), or SGESPAYYTA (SEQ ID NO: 448). In other embodiments, the chimeric polypeptide further comprises an inhibitory moiety, such as a steric inhibitory polypeptide moiety, which can inhibit the action of the interleukin polypeptide. The inhibitory moiety may include, for example, a human serum albumin (HSA) binding domain, or optionally branched or multi-armed polyethylene glycol (PEG). Alternatively, the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof, and an inhibitory moiety, such as a steric inhibitory polypeptide moiety, wherein the inhibitory moiety inhibits the action of the cytokine polypeptide on a cytokine receptor, and in some embodiments, the inhibitory moiety includes a protease-cleavable domain. In some embodiments, inhibition and reduction of cytokine action are achieved simply by attaching an additional domain having a very short linker to the N or C terminus of the interleukin domain. In such embodiments, it is expected that the inhibition will be released by protease digestion of the inhibiting portion or the short linker that tethers the inhibiting substance to the interleukin.Once the domain is broken or released, it can no longer achieve its goal of inhibiting cytokine activity. 【0094】 A pharmaceutical composition, such as a chimeric polypeptide, may contain two or more cytokines, which may be the same cytokine polypeptide or different cytokine polypeptides. For example, two or more different types of cytokines may have complementary functions. In some examples, the first cytokine is IL-2 and the second cytokine is IL-12. In some embodiments, each of the two or more different types of cytokine polypeptides may have a modulating effect on the action of the other cytokine polypeptide. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine polypeptide is T cell activating and the second cytokine polypeptide is T cell non-activating. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine is a chemoattractant, such as CXCL10, and the second cytokine is an immunocytoactivating substance. 【0095】 Preferably, the cytokine polypeptides (including functional fragments) contained in the fusion proteins disclosed herein are not mutated or manipulated to alter the properties of naturally occurring cytokines, including receptor binding affinity and specificity or serum half-life. However, alterations to the amino acid sequence from naturally occurring cytokines (including wild-type) are acceptable, for example, to facilitate cloning and to achieve desired expression levels. 【0096】 blocking part The inhibitory portion may be any portion that inhibits the binding and / or activating ability of a cytokine to its receptor. The inhibitory portion may inhibit the binding and / or activating ability of a cytokine to its receptor by sterically blocking the cytokine and / or by non-covalently binding to it. Examples of suitable inhibitory portions include the full length of the cytokine's homologous receptor or cytokine-binding fragments or mutaines. Antibodies and fragments thereof that bind to cytokines, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies, e.g., heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-type nanobody variable domains (VHH), dAbs, etc., may also be used. Other suitable antigen-binding domains that bind to cytokines may also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticarin, affilin, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of suitable inhibitory polypeptides include polypeptides that sterically inhibit or block the binding of cytokines to their homologous receptors. Beneficially, such portions may also function as half-life extenders. For example, peptides modified by conjugation with water-soluble polymers such as PEG may sterically inhibit or interfere with the binding of cytokines to their receptors. Polypeptides or fragments thereof with long serum half-lives, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, and fragments and mutains of such polypeptides may also be used. For example, antibody and antigen-binding domains that bind to proteins such as HSA, immunoglobulins, or transferrin, which have long serum half-lives, or to receptors such as FcRn or transferrin receptors that are recycled on the plasma membrane, can also inhibit cytokines, specifically when they are bound to those antigens.Examples of such antigen-binding polypeptides include single-chain variable fragments (scFv), single-domain antibodies such as heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-type nanobody variable domains (VHH), and dAbs. Other suitable antigen-binding domains that bind to cytokines may also be used, including non-immunoglobulin proteins that mimic the binding properties and / or structure of antibodies, such as anticarin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. 【0097】 In the illustrative examples, when IL-2 is a cytokine in a chimeric polypeptide, the inhibitory portion may be the full length or a fragment of the alpha chain (IL-2Rα) or beta (IL-2Rβ) or gamma chain (IL-2Rγ) of the IL-2 receptor, or mutaine, an anti-IL-2 single-domain antibody (dAb) or scFv, Fab, an anti-CD25 antibody or a fragment thereof, and an anti-HSA dAb or scFv. As further described herein, when an antibody fragment is used to weaken the action of the cytokine polypeptide, the inhibitory portion in the fusion protein may be a single-chain antibody-binding fragment, such as an scFv. The inhibitory portion may also be half of a double-chain antigen-binding fragment, for example, VH-CH1, which associates with a complementary VL-CL on the second polypeptide to form an antibody-binding site that binds to the cytokine polypeptide. 【0098】 In vivo half-life extension factors Preferably, the chimeric polypeptide contains an element that prolongs the in vivo half-life. Extending the in vivo half-life of therapeutic molecules that naturally have short half-lives allows for more acceptable and manageable drug delivery plans without sacrificing efficacy. As used herein, “half-life prolonging element” is a part of a chimeric polypeptide that prolongs the in vivo half-life and improves pK by altering parameters such as its size (to exceed the renal filtration cutoff), shape, hydrodynamic diameter, charge, or absorption, in vivo distribution, metabolism, and elimination. An exemplary method for improving the pK of a polypeptide is by expressing elements in the polypeptide chain that bind to receptors that are recycled to the cell plasma membrane rather than being degraded in lysosomes, such as FcRn receptors and transferrin receptors on endothelial cells. Three proteins, e.g., human IgG, HSA (or fragments), and transferrin, remain present in human serum much longer than would be predicted by their size alone, which is a function of their binding ability to receptors that are recycled rather than degraded in lysosomes. These proteins or fragments thereof that retain FcRn binding affinity are conventionally ligated to other polypeptides to extend their serum half-life. In one embodiment, the half-life extender is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may be directly bound to the pharmaceutical composition or bound via a short linker. Fragments of HSA may also be used. HSA and its fragments can function as both inhibitory moieties and half-life extenders. Human IgG and Fc fragments can perform similar functions. 【0099】 The serum half-life extending element may be an antigen-binding polypeptide that binds to a protein with a long serum half-life, such as serum albumin or transferrin. Examples of such polypeptides include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as antibodies and their fragments including heavy chain variable domains (VH), light chain variable domains (VL), and camel-type nanobody variable domains (VHH), dAb, etc. Other suitable antigen-binding domains include antibody-binding and / or structurally mimicking non-immunoglobulin proteins, such as anticarin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens. 【0100】 Some preferred serum half-life extenders are polypeptides containing complementarity-determining regions (CDRs) and optional non-CDR loops. Beneficially, such serum half-life extenders can extend the serum half-life of cytokines and may also function as cytokine inhibitors (e.g., by steric inhibition, non-covalent interactions, or a combination thereof) and / or as target-directed domains. In some cases, serum half-life extenders are domains derived from immunoglobulin molecules (Ig molecules) or from engineered protein scaffolds that mimic antibody structure and / or binding activity. Ig can be any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of the Ig molecule folds into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute a "complementarity-determining region" (CDR) that determines the antigen-binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions are further subdivided into highly variable regions called complementarity-determining regions (CDRs), which are highly sequence-variable and / or involved in antigen recognition and / or form structure-defined loops, and which contain scattered regions called more conserved framework regions (FRs). Each VH and VL consists of three CDRs and four FRs aligned in the following order from amino acid terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of this disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding portion described herein. The variable domain of the immunoglobulin molecule has several beta strands aligned on two sheets.Both variable domains of immunoglobulin heavy and light chains contain three hypervariable loops or complementarity-determining regions (CDRs). The three CDRs of the V domain (CDR1, CDR2, and CDR3) are clustered at one end of the beta barrel. The CDRs are loops that link the beta strands BC, C'-C'', and FG of the immunoglobulin fold, while the lower loop linking the beta strands AB, CC', C''-D, and EF of the immunoglobulin fold, and the upper loop linking the DE strand of the immunoglobulin fold, are non-CDR loops. In some embodiments of this disclosure, at least several amino acid residues of the constant domain CH1, CH2, or CH3 are part of the “non-CDR loops” of the binding moiety described herein. In some embodiments, the non-CDR loops include one or more of the following: AB, CD, EF, and DE loops of the C1-group domain of Ig or an Ig-like molecule; AB, CC', EF, FG, BC, and EC' loops of the C2-group domain of Ig or an Ig-like molecule; and DE, BD, GF, A(A1A2)B, and EF loops of the I(intermediate)-group domain of Ig or an Ig-like molecule. 【0101】 Within the variable domain, CDRs are thought to be responsible for antigen recognition and binding, while FR residues are considered scaffolds for CDRs. However, in some cases, some FR residues play a crucial role in antigen recognition and binding. Framework region residues that influence Ag binding can be divided into two categories. The first are FR residues that come into contact with the antigen and are therefore part of the binding site, and some of these residues are located near the CDR. Other residues are located away from the CDR but are very close to it in the three-dimensional structure of the molecule, for example, in the heavy chain loop. 【0102】 The binding moiety is any type of polypeptide. For example, in some cases the binding moiety is a native peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered giant serum protein. Giant serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolds (as proposed in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffolds, protein G-associated albumin-binding modules, or DNA or RNA aptamer scaffolds. 【0103】 In some cases, the serum half-life extension element includes a binding site for giant serum proteins. In some embodiments, the CDR provides a binding site for giant serum proteins. Giant serum proteins are, in some examples, globulins, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomers, factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDR forms a binding site for immunoglobulin light chains, such as Igκ-free light chains or Igλ-free light chains. 【0104】 The serum half-life extension component may be any type of binding domain, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding portion is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a camel-derived nanobody variable domain (VHH). In other embodiments, the binding portion is a non-Ig binding domain, i.e., an antibody mimetic, e.g., antikalin, affilin, affibody molecule, affimer, afitin, alpha-body, avimer, DARPin, finomer, kunitz domain peptide, and monobody. 【0105】 In other embodiments, the serum half-life extender may be a water-soluble polymer or a peptide conjugated to a water-soluble polymer, such as PEG. As used herein, “PEG,” “polyethylene glycol,” and “poly(ethylene glycol)” are interchangeable and encompass any non-peptide water-soluble poly(ethylene oxide). The term “PEG” also means a polymer containing a majority, i.e., more than 50%, of the –OCH2CH2– repeating subunits. With respect to a particular form, PEG can take any number of different molecular weights, as well as structural or geometric configurations, such as “branched,” “linear,” “fork-type,” “polyfunctional,” etc., which are described in more detail below. PEG is not limited to a particular structure and can be linear (e.g., end-bound, e.g., alkoxyPEG, or bifunctional PEG), branched or polyfunctional (e.g., fork-type PEG, or PEG conjugated to a polyol core), or dendrimer (or star-shaped) structure, each of which may or may not have one or more degradable linkages. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns, and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating trippolymers, random trippolymers, and block trippolymers. PEG can be conjugated to polypeptides and peptides by any suitable method. Typically, a peptide or polypeptide containing amino acids having side chains containing amines, sulfhydryls, carboxylic acids, or hydroxyl functional groups, such as cysteine, lysine, asparagine, glutamine, threonine, triosine, serine, aspartic acid, and glutamic acid, is reacted with a reactive PEG derivative, such as N-hydroxysuccinamidyl ester PEG. 【0106】 Target-directed and retention domains For certain applications, it may be desirable to maximize the amount of time a construct spends in its desired location within the body. This can be achieved by incorporating an additional domain into a chimeric polypeptide (fusion protein) to influence its movement within the body. For example, a chimeric nucleic acid may encode a domain that directs the polypeptide to a location in the body, e.g., tumor cells or inflammatory sites; this domain is referred to as the “target-directing domain,” and / or a domain that encodes a domain that retains the polypeptide in the body, e.g., tumor cells or inflammatory sites; this domain is referred to as the “retention domain.” In some embodiments, a domain may function as both a target-directing and a retention domain. In some embodiments, the target-directing domain and / or retention domain is specific to a protease-rich environment. In some embodiments, the encoded target-directing domain and / or retention domain is specific to regulatory T cells (Tregs), e.g., targeting CCR4 or CD39 receptors. Other suitable target-directing and / or retention domains include those having homologous ligands that are overexpressed in inflammatory tissue, e.g., IL-1 receptors or IL-6 receptors. In other embodiments, preferred target-directing and / or retention domains include those having a homologous ligand overexpressed in tumor tissue, such as Epcam, CEA, or mesothelin. In some embodiments, the target-directing domain is linked to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammation or cancer-specific protease) to release the interleukin's full action at a desired site. In some embodiments, the target-directing and / or retention domain is linked to the interleukin via a linker that is not cleaved at the site of action (e.g., by an inflammation or cancer-specific protease) and allows the cytokine to remain at a desired site. 【0107】 In some cases, the optimal antigen is expressed on the surface of diseased cells or tissues, such as tumor or cancer cells. Antigens that are useful for targeting and retaining tumors include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins that contain two target-directing and / or retention domains that bind to two different target antigens known to be expressed on diseased cells or tissues. Exemplary antigen-binding domain pairs include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3. 【0108】 Suitable target-directed and / or retaining domains include antigen-binding domains, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies, such as heavy-chain variable domains (VH), light-chain variable domains (VL), and camel-type nanobody variable domains (VHH), antibodies and their fragments, such as dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticarin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, finomers, kunitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens. 【0109】 In some embodiments, the target-directed and / or retaining domain specifically binds to cell surface molecules. In some embodiments, the target-directed and / or retaining domain specifically binds to tumor antigens. In some embodiments, the target-directed polypeptide specifically and independently binds to tumor antigens selected from at least one of fibroblast-activating protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling molecule 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide specifically and independently binds to two different antigens, at least one of which is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. 【0110】 Target-directed and / or retained antigens may be tumor antigens expressed on tumor cells. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 Examples include E7, ITGA2, ITGA3, SLC39A6, MAGE, Mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-Cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1. 【0111】 Target-directed and / or retained antigens may be immune checkpoint proteins. Examples of immune checkpoint proteins include, but are not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA. 【0112】 Target-directed and / or retaining antigens may be cell surface molecules, such as proteins, lipids, or polysaccharides. In some embodiments, target-directed and / or retaining antigens are located on tumor cells, virus-infected cells, bacterial-infected cells, damaged erythrocytes, arterial plaque cells, or inflammatory or fibrous tissue cells. Target-directed and / or retaining antigens may include immune response modifiers. Examples of immune response modifiers include, but are not limited to, granulocyte-macrophage-colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR. 【0113】 Target-directed and / or retained antigens can be cytokine receptors. Examples of cytokine receptors include type I cytokine receptors, e.g., GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor; type II cytokine receptors, e.g., IFN-alpha receptors (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptors (IFNGR1, IFNGR2), type II IL receptor; and chemokine receptors, e.g., CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, XC chemokine receptor. Examples of receptors include, but are not limited to, mokine receptors; tumor necrosis receptor superfamily receptors, e.g., TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, e.g., TGF-beta receptor 1, TGF-beta receptor 2; and Ig superfamily receptors, e.g., IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR. 【0114】 Linker As described above, the pharmaceutical composition comprises one or more linker sequences. The linker sequences help to provide flexibility between polypeptides, for example, so that the inhibitory portion can inhibit the action of the cytokine polypeptide. The linker sequences may be located between any or all of the cytokine polypeptide, serum half-life extender, and / or inhibitory portion. As described herein, at least one of the linkers is protease-cleavable and contains one or more cleavage sites for one or more desired proteases. Preferably, the desired proteases are abundant or selectively expressed at the desired cytokine action site (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the desired cytokine action site. 【0115】 Suitable linkers can vary in length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, for example, from 4 amino acids to 10 amino acids, from 1 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. 【0116】 It is recognized that the orientation of components in a pharmaceutical composition is generally a matter of structural selection, and that multiple orientations are possible and all are intended to be covered by this disclosure. For example, the inhibitory moiety may be located at the C-terminus or N-terminus of a cytokine polypeptide. 【0117】 Proteases known to be associated with diseased cells or tissues include serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, C, D, E, K, L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelicin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidine, bromelain, calpain, caspases, caspases-3, and Mir Examples include, but are not limited to, l-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, plasmmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequence provided herein may be selected from, for example, the group consisting of prostate-specific antigen (PSA), matrix metalloproteinase (MMP), disintegrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP may be, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9). 【0118】 Table 1 shows proteases useful in the methods disclosed herein, and Table 1a shows exemplary proteases and their cleavage sites: [Table 1-1] [Table 1-2] [Table 1a-1] [Table 1a-2] [Table 1a-3] [Table 1a-4] 【0119】 This specification provides pharmaceutical compositions comprising polypeptide sequences. With respect to any peptide, polypeptide, and protein, including fragments, it is understood that further modifications may occur in the amino acid sequence (amino acid sequence variant) of a chimeric polypeptide, without altering the properties or function of the peptide, polypeptide, or protein. Such modifications include conservative amino acid substitutions, which are described in more detail below. 【0120】 The compositions provided herein have desired functions. Each composition comprises at least a cytokine polypeptide, e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFNa or IFNγ, or a chemokine, e.g., CXCL10, CCL19, CCL20, CCL21, an inhibitory moiety, e.g., a steric inhibitory polypeptide, and an optional serum half-life extender, and an optional target-directed polypeptide, wherein one or more linkers link each polypeptide in the composition. A first polypeptide, e.g., IL-2 mutein, is provided to be an active agent. An inhibitory moiety is provided to inhibit the action of an interleukin. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed at the intended target of the active agent. Optionally, the inhibitory moiety inhibits the action of the first polypeptide by binding to an interleukin polypeptide. In some embodiments, the inhibitory portion, such as a steric inhibitory peptide, is linked to the interleukin via a protease-cleavable linker that cleaves at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full action of the cytokine at the desired site. 【0121】 Protease cleavage sites may be naturally occurring or artificially engineered. Artificially engineered protease cleavage sites may be cleaved by a desired environment in which cleavage occurs, such as by more than one tumor-specific proteases. Protease cleavage sites may be capable of being cleaved by at least one protease, at least two proteases, at least three proteases, or at least four proteases. 【0122】 In some embodiments, the linker is glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser) n (Sequence ID 443) or (Gly3Ser) nThis includes (SEQ ID NO: 444), where n is 1, 2, 3, 4, or 5. In one embodiment, the sortase recognition motif includes the peptide sequence LPXTG (SEQ ID NO: 442), where X is any amino acid. In one embodiment, there is a covalent linkage between a reactive lysine residue bound to the C-terminus of the cytokine polypeptide and a reactive aspartate bound to the N-terminus of the inhibition moiety or other moiety. In one embodiment, the covalent linkage is between a reactive aspartate residue bound to the N-terminus of the cytokine polypeptide and a reactive lysine residue bound to the C-terminus of the inhibition moiety or other moiety. 【0123】 Cutting and induction possibilities As described herein, the action of cytokine polypeptides in the context of fusion proteins is attenuated, and protease cleavage at the desired site of action, e.g., in the tumor microenvironment, releases from the fusion protein a form of cytokine that is far more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine receptor activating (agonist) action of a fusion polypeptide may be about 1 / 10, 1 / 50, 1 / 100, 1 / 250, 1 / 500, or 1 / 1000 of the cytokine receptor activating action of a cytokine polypeptide as a separate molecular entity. A cytokine polypeptide that is part of a fusion protein exists as a separate molecular entity if it contains amino acids that are substantially identical to those of the cytokine polypeptide, substantially no additional amino acids, and is not associated with other molecules (by covalent or non-covalent bonds). If necessary, the cytokine polypeptide as a separate molecular entity may contain several additional amino acid sequences, e.g., tags or short sequences useful for expression and / or purification. 【0124】 In other examples, the cytokine receptor activating (agonist) effect of fusion polypeptides is approximately 1 / 10, 1 / 50, 1 / 100, 1 / 250, 1 / 500, or 1 / 1000 of the cytokine receptor activating effect of polypeptides containing cytokine polypeptides produced by cleavage of protease-cleavable linkers in the fusion protein. In other words, the cytokine receptor activating (agonist) effect of polypeptides containing cytokine polypeptides produced by cleavage of protease-cleavable linkers in the fusion protein is at least approximately 10 times, at least approximately 50 times, at least approximately 100 times, at least approximately 250 times, at least approximately 500 times, or at least approximately 1000 times greater than the cytokine receptor activating effect of fusion proteins. 【0125】 Polypeptide variants and amino acid substitutions The polypeptides described herein may include components (e.g., cytokines, inhibitory moieties) that have the same amino acid sequence as the corresponding naturally occurring proteins (e.g., IL-2, IL-15, HSA) or that have a different amino acid sequence from the naturally occurring proteins, as long as the desired function is maintained. It is understood that one way of defining any known modifications and derivatives or potential occurrences of the proteins and nucleic acids encoding them herein is by defining sequence variants with respect to identity with a particular known reference sequence. Specifically, polypeptides and nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity with the chimeric polypeptides provided herein. For example, polypeptides or nucleic acids are provided that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99 percent identity with any of the nucleic acid or polypeptide sequences described herein. Those skilled in the art will readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, identity can be calculated after aligning the two sequences such that their identity is at its highest level. 【0126】 Another method for calculating identity can be performed by publicly available algorithms. Optimal alignment of sequences for comparison can be achieved by the local identity algorithm of Smith and Waterman, Adv.Appl.Math.2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection. 【0127】 Identification of nucleic acids of the same kind can be obtained by algorithms disclosed, for example, Zuker, Science 244:48-52 (1989), Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989), and Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference, at least with respect to data relating to nucleic acid alignment. It is understood that any of these methods may be typically used, and the results of these various methods may differ in some cases, but if identity is found by at least one of these methods, then the sequence can be said to have the identity of the designation and be disclosed herein. 【0128】 Protein modification includes amino acid sequence modification. Amino acid sequence modifications can occur naturally as allele mutations (e.g., due to genetic polymorphism), due to environmental influences (e.g., UV exposure), or through human intervention (e.g., mutagenesis of clonal DNA sequences) such as induced point, deletion, insertion, and substitution mutations. These modifications can result in changes to the amino acid sequence, silent mutations, changes to restriction sites, or other specific mutations. Amino acid sequence modifications typically fall into one or more of three classes: substitution, insertion, or deletion modifications. Insertions include amino and / or carboxyl-terminus fusions, as well as intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than amino or carboxyl-terminus fusions, for example, around 1-4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, this involves the deletion of approximately 2-6 residues or less at any single site in the protein molecule. Amino acid substitutions are typically single-residue substitutions, but can occur at multiple different locations simultaneously; insertions are usually around 1 to 10 amino acid residues; and deletions range from approximately 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., two-residue deletions or two-residue insertions. Substitutions, deletions, insertions, or any combination thereof can be combined to obtain the final construct. Mutations should not place the sequence outside the reading frame and, preferably, should not create complementary regions that may give rise to secondary mRNA structures. A substitution modification involves the removal of at least one residue and the insertion of a different residue in its place. Such substitutions are generally made according to Table 2 below and are referred to as conservative substitutions. [Table 2] 【0129】 Modifications are carried out by known methods, including the specific amino acid substitutions mentioned. For example, modifications are made by site-specific mutagenesis of nucleotides in the polypeptide-encoding DNA to generate DNA encoding the modification, and then expressing the DNA in recombinant cell culture. Techniques for inducing substitutional mutations at specific sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutagenesis. 【0130】 Modifications may be selected to optimize binding affinity. For example, the binding affinity of scFv can be altered by introducing random mutations within the complementarity-determining region (CDR) using affinity maturation techniques. Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens, or error-prone PCR. Multiple mutations and selections may be performed, for example, using phage display. 【0131】 This disclosure also relates to nucleic acids encoding the chimeric polypeptides described herein, as well as the use of such nucleic acids for the production of chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) encoding chimeric polypeptides, as well as the therapeutic use of such DNA and RNA molecules. 【0132】 Exemplary composition The exemplary fusion proteins of the present invention combine the above elements in various orientations. The orientations described in this section are intended as examples only and should not be considered limiting. 【0133】 In some embodiments, the fusion protein includes a cytokine, an inhibitory moiety, and a half-life extension element. In some embodiments, the cytokine is located between the half-life extension element and the inhibitory moiety. In some embodiments, the cytokine is located on the N-terminal side of the inhibitory moiety and the half-life extension element. In some such embodiments, the cytokine is located near the inhibitory moiety; in some such embodiments, the cytokine is located near the half-life extension element. In any embodiment, at least one protease-cleavable linker must be included so that the cytokine can be activated by cleavage. In some embodiments, the cytokine is located on the C-terminal side of the inhibitory moiety and the half-life extension element. Additional elements may be linked by a cleavable linker, a non-cleavable linker, or direct fusion. 【0134】 In some embodiments, the inhibitory domains used can have an extended half-life, and the cytokine is positioned between two such inhibitory domains. In some embodiments, the cytokine is positioned between two inhibitory domains, one of which can have an extended half-life. 【0135】 In some embodiments, two cytokines are included in the same construct. In some embodiments, each cytokine is ligated to two inhibitory domains (three in total per molecule), with one inhibitory domain located between the two cytokine domains. In some embodiments, one or more additional half-life extension domains may be included to optimize pharmacokinetic properties. In some cases, it is beneficial to include two identical cytokines to facilitate dimerization. An example of a cytokine that acts as a dimer is IFN. 【0136】 In some embodiments, three cytokines are included in the same construct. In some embodiments, the third cytokine may function to block the other two, instead of blocking the blocking domain between the two cytokines. 【0137】 Preferred half-life-extending elements for use in the fusion protein are human serum albumin (HSA), an antibody or antibody fragment that binds to serum albumin (e.g., scFv, dAb), human or humanized IgG, or any of the above fragments. In some preferred embodiments, the inhibitory moiety is human serum albumin (HSA), or an antibody or antibody fragment that binds to serum albumin, an antibody that binds to a cytokine and prevents the binding activation or reactivation of the cytokine receptor, another cytokine, or any of the above fragments. In preferred embodiments including an additional target-directing domain, the target-directing domain is an antibody that binds to cell surface proteins abundant on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin. 【0138】 In embodiments, the fusion protein may contain an IL-2 polypeptide. A fusion protein containing an IL-2 polypeptide may contain or consist of one of the amino acid sequences of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646. The fusion proteins disclosed as SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608 and 636-646 are referred to herein as ACP289-ACP292, ACP296-ACP302, WW0301, ACP304-ACP306, ACP309-ACP313, WW03 These are also referred to as 53, ACP414, ACP336~ACP398, WW0472~WW0477, ACP406~ACP426, ACP439~ACP447, ACP451~ACP471, WW0729, WW0734~WW0792, ACP101, ACP293~ACP295, ACP316~ACP335, ACP427~ACP438, and ACP448~ACP450. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 272. The fusion protein may contain the amino acid sequence of SEQ ID NO: 286. The fusion protein may contain the amino acid sequence of SEQ ID NO: 362. The fusion protein may contain the amino acid sequence of SEQ ID NO: 336. The fusion protein may contain the amino acid sequence of SEQ ID NO: 348. The fusion protein may contain the amino acid sequence of SEQ ID NO: 363. The fusion protein may contain the amino acid sequence of SEQ ID NO: 580. 【0139】 In embodiments, the fusion protein may contain an IL-12 polypeptide. A fusion protein containing an IL-12 polypeptide may contain or consist of one of the amino acid sequences of SEQ ID NOs. 368-371, 434-440, 453-519, or 523-538. The fusion proteins disclosed as SEQ ID NOs. 368-371, 434-440, 453-519, or 523-538 are referred to herein as ACP240-ACP245, ACP247, ACP285-ACP288, WW0641, WW0649-WW0652, WW0662-WW0725, WW0765-WW0772, and WW0796-WW0803. For example, the fusion protein may contain the amino acid sequence of SEQ ID NO: 459. The fusion protein may contain the amino acid sequence of SEQ ID NO: 466. The fusion protein may contain the amino acid sequence of SEQ ID NO: 484. The fusion protein may contain the amino acid sequence of SEQ ID NO: 506. 【0140】 In embodiments, the fusion protein contains an IFN (e.g., IFN gamma, IFN alpha, IFN beta) polypeptide. In some examples, the IFN polypeptide is IFN alpha or IFN beta. A fusion protein containing an IFN polypeptide may contain or consist of the amino acid sequences of SEQ ID NOs. 421-430 and 539-578. The fusion proteins disclosed as SEQ ID NOs. 421-430 and 539-578 may be referred to herein as ACP200-ACP209, WW0644-WW0648, WW0781-WW0786, WW0815-WW0822, WW0831-WW0834, WW0737-WW0748 and WW0787-WW0790. For example, the fusion protein may contain the amino acid sequence of SEQ ID NOs. 421. The fusion protein may contain the amino acid sequence of SEQ ID NOs. 428. The fusion protein may contain the amino acid sequence of SEQ ID NOs. 541. The fusion protein may contain the amino acid sequence of SEQ ID NO: 558. The fusion protein may contain the amino acid sequence of SEQ ID NO: 577. 【0141】 In some embodiments, the fusion polypeptides disclosed herein may be covalently or noncovalently bound to a second polypeptide chain. For example, the fusion polypeptide may dimerize (i.e., form a dimer), or a portion of the fusion polypeptide may associate with another polypeptide, resulting in the formation of a functional binding site for, for example, a cytokine polypeptide or serum albumin. In some embodiments, the second polypeptide chain and the inhibitory portion on the fusion polypeptide are complementary and together form a functional binding site having specificity for the cytokine polypeptide contained in the fusion polypeptide. Exemplary functional binding sites that may be formed by an inhibitory portion of a fusion polypeptide and a complementary second polypeptide include antigen-binding sites of antibodies, such as the Fab fragment or a portion thereof of an antibody. For example, one chain of Fab that binds to a cytokine may be the inhibitory portion of the fusion polypeptide, e.g., VH-CH1, and the complementary VL-CL may be part of the second polypeptide. In such cases, the inhibitory portion of the fusion protein, namely VH-CH1, and the complementary second polypeptide containing VL-CL may associate to form a functional binding site with specificity for cytokine polypeptides (e.g., IL-2, IL-12, IFN-alpha, IFN-beta) contained in the fusion protein, thereby weakening the cytokine polypeptide activity. At least a portion of the inhibitory portion may be on the second polypeptide chain and may include at least a portion of the inhibitory portion that associates with the inhibitory portion on the fusion polypeptide. 【0142】 In embodiments, the fusion protein containing the IL-2 cytokine polypeptide may be covalently or noncovalently bound to the second polypeptide chain. The second polypeptide chain may contain an antibody light chain VL-CL comprising or consisting of the amino acid sequence of SEQ ID NOs. 263, 264, or 333. Such a second polypeptide may be bound to a complementary VH-CH1 polypeptide contained in the fusion protein, for example, in SEQ ID NOs. 362, 363, 325, 286, 579, 581, or 582. The second polypeptide chains disclosed as SEQ ID NOs. 263, 264, and 333 may be referred to herein as WW0523(ACP381), WW0524(ACP382), or WW0556(ACP414). 【0143】 In the embodiment, the fusion polypeptide may or may consist of the amino acid sequence of SEQ ID NOs: 362, 363, 325, 286, 579, 581, or 582, and the second polypeptide chain may or may consist of the amino acid sequence of SEQ ID NOs: 263, 264, or 333. The fusion polypeptides disclosed as SEQ ID NOs. 362, 363, 325, 286, 579, 581, or 582 may be designated as WW0520(ACP378), WW0521(ACP379), WW0548(ACP406), WW0621(ACP457), WW0729, WW0735, or WW0736, and the second polypeptide chains disclosed as SEQ ID NOs. 263, 264, and 333 may be designated herein as WW0523(ACP381), WW0524(ACP382), or WW0556(ACP414). 【0144】 For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264.For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 286, and the second polypeptide may contain or be derived from the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may contain or be derived from the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may contain or be derived from the amino acid sequence of SEQ ID NO: 233. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 263. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 264. For example, the fusion protein may contain or be derived from SEQ ID NO: 581, and the second polypeptide chain may contain or be derived from SEQ ID NO: 333. For example, the fusion protein may contain or be derived from SEQ ID NO: 582, and the second polypeptide chain may contain or be derived from SEQ ID NO: 263. For example, the fusion protein may contain or be derived from SEQ ID NO: 582, and the second polypeptide chain may contain or be derived from SEQ ID NO: 264. For example, the fusion protein may contain or be derived from SEQ ID NO: 582, and the second polypeptide chain may contain or be derived from SEQ ID NO: 333. 【0145】 Treatment method and pharmaceutical composition This disclosure also relates to pharmaceutical compositions comprising one or more fusion proteins disclosed herein in optional combination with another therapeutic agent, preferably an immunomodulator or an anticancer agent. This disclosure also relates to the use of such pharmaceutical compositions in the treatment of cancer and the use of one or more fusion proteins in optional combination with another therapeutic agent. 【0146】 The therapeutic combinations disclosed herein may include, for example, a fusion protein containing an IL-2 polypeptide, a fusion protein containing an IL-12 polypeptide, or a fusion protein containing an IFN polypeptide. Therapies may be provided using two or more fusion proteins. For example, a therapeutic combination may include a fusion protein containing an IL-2 polypeptide and a fusion protein containing an IL-12 polypeptide, a fusion protein containing an IL-2 polypeptide and a fusion protein containing an IFN polypeptide, or a fusion protein containing an IL-12 polypeptide and an IFN polypeptide. 【0147】 The therapeutic combinations disclosed herein may comprise a fusion polypeptide cytokine polypeptide [A], an inhibitory moiety [D], an optional half-life extension moiety [H], and a protease-cleavable polypeptide linker, wherein the cytokine polypeptide, the inhibitory moiety, and the optional half-life extension element (if present) are functionally linked by the protease-cleavable polypeptide linker, the fusion polypeptide having a weakened cytokine receptor activating effect, the cytokine receptor activating effect of the fusion polypeptide being about one-tenth or less of the cytokine receptor activating effect of a polypeptide containing the cytokine polypeptide generated by cleavage of the protease-cleavable linker, and the fusion polypeptide is of formula: [A]-[L1]-[H]-[L2]-[D](I); [D]-[L2]-[H]-[L1]-[A](II); [A]-[L1]-[D]-[L2]-[H](III); [H]-[L2]-[D]-[L1]-[A](IV); [H]-[L1]-[A]-[L2']-[D](V); [D]-[L1]-[A]-[L2']-[H](VI); [In the formula, [A] is a cytokine polypeptide, [D] is an inhibitory region, [H] is a half-life extension region, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that can be selectively protease-cleaved, and [L2'] is a protease-cleavable polypeptide linker.] The therapeutic composition may comprise a second fusion polypeptide comprising at least one each of a second cytokine polypeptide [A], an inhibitory moiety [D], an optional half-life extender [H], and a protease-cleavable polypeptide linker [L], wherein the cytokine polypeptide, the cytokine inhibitory moiety, and the optional half-life extender (if present) are functionally linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has a weakened cytokine receptor activating effect, the cytokine receptor activating effect of the fusion polypeptide being about one-tenth or less of the cytokine receptor activating effect of a polypeptide containing a cytokine polypeptide generated by cleavage of the protease-cleavable linker. The second fusion polypeptide has the formula: [A]-[L1]-[H]-[L2]-[D](I); [D]-[L2]-[H]-[L1]-[A](II); [A]-[L1]-[D]-[L2]-[H](III); [H]-[L2]-[D]-[L1]-[A](IV); [H]-[L1]-[A]-[L2']-[D](V); [D]-[L1]-[A]-[L2']-[H](VI); [In the formula, [A] is a cytokine polypeptide, [D] is an inhibitory region, [H] is a half-life extension region, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that can be selectively protease-cleaved, and [L2'] is a protease-cleavable polypeptide linker.] It may have. 【0148】 The therapeutic combination drugs disclosed herein are first fusion proteins comprising amino acids selected from the group consisting of SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430 and 539-578. The first fusion protein may also contain a second fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578. It is preferable that the first fusion protein and the second fusion protein are different. In some preferred embodiments, the therapeutic combination comprises a first fusion protein containing an amino acid sequence selected from SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646, and a second fusion protein containing an amino acid sequence selected from SEQ ID NOs: 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578. In some preferred embodiments, the therapeutic combination comprises a first fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 368-371, 434-440, 453-519 or 523-538, and a second fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 636-646, 579-608, 421-430 and 539-578.In some preferred embodiments, the therapeutic combination comprises a first fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 421-430 and 539-578, and a second fusion protein comprising an amino acid sequence selected from SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538 or a combination thereof. 【0149】 The therapeutic combinations disclosed herein may comprise a second polypeptide chain and a first fusion protein covalently or noncovalently bonded to the therapeutic agent. The therapeutic combination drug may comprise (i) a fusion polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 362, 363, 325, 286, 579, 581, or 582, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NOs: 263, 264, or 333, and (ii) a second therapeutic agent, the second therapeutic agent being a second fusion polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, 539-578, or combinations thereof. It is preferable that the first fusion protein and the second fusion protein are not the same. 【0150】 In embodiments, the therapeutic combination may include additional therapeutic agents (e.g., one, two, three, four, five or more therapeutic agents). In embodiments, the therapeutic combination may include two or more fusion proteins and one or more therapeutic agents, preferably agents for treating cancer. 【0151】 Other exemplary therapeutic agents include, but are not limited to, chemomancyns (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2, VEGF inhibitors), antibody-drug conjugates, cell therapies (e.g., CAR-T, T-cell therapies), oncolytic viruses, radiotherapy, and / or small molecules. 【0152】 Non-exclusive examples of anticancer drugs that may be used include: asibicin, acralubicin, acodazole hydrochloride, acronin, adzeresin, aldesleukin, altretamine, ambomycin, amethantrone acetate, aminoglutethimide, amsacrin, anastrozole, anthramycin, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisanthren hydrochloride, bisnafide dimesylate, bizeresin, bleoma Isin sulfate, Brequinal sodium, Bropyrimin, Busulfan, Kactinomycin, Carsterone, Calasemide, Carvetimer, Carboplatin, Carmustine, Carbicin hydrochloride, Carzeresin, Sedefingol, Chlorambucil, Cyloremycin, Cisplatin, Cladribine, Cristonol mesylate, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Daunorubicin hydrochloride, Decitabine, Dexormaplatin, Dezaguanine, Dezaguani mesylate Diazyquinone, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, erusamitrusine, enloplatin, empromart, epipropidine, epirubicin hydrochloride, erbrozol, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, Fadrozol hydrochloride, fazarabine, fenretinide, floxyuridine, fludarabine phosphate, fluorouracil, fluorocitabine, fosquidone, fostriesin sodium salt, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, irmofosin, interleukin II (including recombinant interleukin II or rIL2), interferon alpha-2a, interferon alpha-2b, interferon alpha-n1Interferon alpha-n3, interferon beta-Ia, interferon gamma-Ib, iproplatin, irinotecan hydrochloride, lanreotide acetate, letrozole, leuprolide acetate, rialozol hydrochloride, lometrexol sodium salt, lomustine, loxoxantrone hydrochloride, masopropyl, meitansine, mechloretamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogalyl, mercaptopurine, methotrexate, methotrexate sodium salt, metoprin, metsuredepa, mitind Mid, Mitocalcin, Mitochromin, Mitogiline, Mitomarcin, Mitospel, Mitotan, Mitoxantrone hydrochloride, Mycophenolic acid, Nocodazole, Nogaramycin, Ormaplatin, Oxythran, Paclitaxel, Pegasparagaze, Periomycin, Pentamustine, Peplomycin sulfate, Perphosphamide, Pipobroman, Piposulfan, Piroxantrone hydrochloride, Plicamycin, Promethan, Porfimer sodium, Porphyromycin, Prednimustine, Procarbazine hydrochloride, Puroma Icin, puromycin hydrochloride, pyrazofurin, ribopurine, logretimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium salt, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, slofenucle, tarisomycin, tecogalan sodium salt, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxylone, testactone, thiamiprine, thioguanine, thiotepa, thiazophrine, tirapaza Examples include methyl phosphate, toremifene citrate, trestron acetate, trisilibine phosphate, trimethrexate, trimethrexate glucuronide, triptorelin, tubrosol hydrochloride, uracil mustard, uredepa, bapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindestine sulfate, vinepidine sulfate, vingricinate sulfate, vinoleulosine sulfate, vinorelbine tartrate, vinzolidine sulfate, vinzolidine sulfate, borozol, zeniplatin, dinostatin, and zolubicin hydrochloride. 【0153】 Accordingly, this disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with chemotherapeutic agents, such as Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisanthren, Noantrone, Thiguanine, Cytaribine, and Procarabizine. This disclosure also relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with antibody-drug conjugates. Various antibody-drug conjugates suitable for use in cancer therapy are well known and typically include antibodies that bind to cellular antigens that are preferentially expressed or expressed at high levels on tumor cells, and cytotoxic agents. This disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with cell therapy, such as CAR-T or T cell therapy. This disclosure also relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with oncolytic viruses. Exemplary oncolytic viruses include oncolytic adenovirus, herpes simplex virus type 1 (HSV), poliovirus, measles virus (MV), Newcastle disease virus (NDV), reovirus, varicella stomatitis virus (VSV), and Zika virus. This disclosure also relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with radiotherapy, such as external beam or internal radiotherapy. 【0154】 This disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) in combination with cytokines (e.g., IL-2, IL-15), signal-inducing inhibitors (e.g., BRAF inhibitors or MEK inhibitors), checkpoint inhibitors (e.g., PDL-1, PD-1, CTLA-4), c-met inhibitors, kinase inhibitors (e.g., VGEF inhibitors), proteasome inhibitors, mTOR inhibitors, and angiogenesis inhibitors. In embodiments, any one of the fusion proteins disclosed herein (e.g., fusion proteins comprising IL-2 polypeptide and IL-12 polypeptide or IFN polypeptide) may be combined with an anti-PD-L1 agent or an anti-PD-1 agent. Examples of PD-1 and / or PD-L1 inhibitors include, but are not limited to, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, INCMGA00012, AMP-224, and AMP-514. In embodiments, fusion proteins comprising amino acid sequences selected from SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578 may be combined with checkpoint inhibitors, such as PDL-1, PD-1, or CTL-4. 【0155】 Furthermore, methods are provided for treating subjects who have or are at risk of developing a disease or disorder, such as proliferative disorders, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, or graft-versus-host diseases. The methods disclosed herein are preferably used to treat subjects having cancer. A method for administering an effective amount of the fusion protein disclosed herein, which is typically administered as a pharmaceutical composition, to a subject in need thereof. In some embodiments, the method further includes selecting a subject who has or is at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises an inhibited cytokine, its fragment, variant, subunit, or mutein, which is activated at the site of inflammation or tumor. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment or mutein, and a serum half-life extending element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its variant, subunit, fragment or mutein, and an inhibitory moiety, such as a steric inhibitory polypeptide, which can sterically inhibit the action of the cytokine polypeptide, its fragment or mutein. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutain thereof, an inhibitory moiety, and a serum half-life extension element. 【0156】 Inflammation is part of a complex biological response of body tissues to harmful stimuli, such as pathogens, damaging cells, or irritants, and is a defensive response involving immune cells, blood vessels, and molecular signaling molecules. The function of inflammation is to eliminate the initial cause of cellular damage, remove damaged necrotic cells and tissues from the initial injury and inflammatory process, and initiate tissue repair. Inflammation can arise from infection, symptoms, or diseases, such as cancer, atherosclerosis, allergies, myopathy, HIV, obesity, or autoimmune diseases. Autoimmune diseases are chronic conditions resulting from an abnormal immune response to autoantigens. Autoimmune diseases that may be treated with polypeptides disclosed herein include, but are not limited to, lupus, celiac disease, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus. 【0157】 The pharmaceutical composition may contain one or more protease-cleavable linker sequences. The linker sequences help to provide flexibility between polypeptides, allowing each polypeptide to inhibit the action of the first polypeptide. The linker sequences may be located between any or all of the cytokine polypeptide, its fragment or mutain, the inhibitory moiety, and the serum half-life extension element. Optionally, the composition may contain two, three, four, or five linker sequences. The linker sequences, two, three, or four linker sequences, may be the same or different. In one embodiment, the linker sequence includes GGGGS (SEQ ID NO: 449), GSGSGS (SEQ ID NO: 450), or G(SGGG)2SGGT (SEQ ID NO: 451). In another embodiment, the linker includes a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 445), IPVSLRSG (SEQ ID NO: 446), VPLSLYSG (SEQ ID NO: 447), and SGESPAYYTA (SEQ ID NO: 448). 【0158】 In some embodiments, the linker is cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, or tumor cell surface proteases. 【0159】 Suitable linkers can vary in length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, for example, from 4 amino acids to 10 amino acids, from 1 amino acids to 9 amino acids, from 6 amino acids to 8 amino acids, or from 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. 【0160】 Furthermore, a method is provided for treating subjects who have cancer or are at risk of developing it. The method comprises administering an effective amount of the chimeric polypeptide (fusion protein) described herein, which is typically administered as a pharmaceutical composition, to a subject in need thereof. In some embodiments, the method further comprises selecting a subject who has cancer or is at risk of developing it. The pharmaceutical composition preferably comprises an inhibited cytokine, a fragment thereof, or a mutain that is activated at the tumor site. 【0161】 The methods disclosed herein may be used to treat any suitable cancer, including hematopoietic malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Suitable cancers exemplified include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendiceal cancer, astrocytoma, basal cell carcinoma, brain tumor, cholangiocarcinoma, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary origin, cardiac tumor, cervical cancer, chondroma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonic tumor, embryonic carcinoma, ependymal cell tumor, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, ocular cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioblastoma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, non-invasive lobular carcinoma, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, Lanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell carcinoma of unknown primary origin, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, chromophilia Examples include sex cell tumors, pituitary tumors, pleuropulmonary blastomas, primary central nervous system lymphomas, prostate cancer, kidney cancer, renal cell carcinoma, renal pelvis and ureteral cancers, retinoblastoma, rhabdoid tumors, salivary gland cancers, Sézary syndrome, skin cancers, small cell lung cancers, small intestine cancers, soft tissue sarcomas, spinal cord tumors, gastric cancers, T-cell lymphomas, teratomas, testicular cancers, pharyngeal cancers, thymomas and thymic carcinomas, thyroid cancers, urethral cancers, uterine cancers, vaginal cancers, vulvar cancers, and Wilms' tumors. 【0162】 Preferably, the tumor is a solid tumor. Colon cancer, lung cancer, melanoma, sarcoma, renal cell carcinoma, and breast cancer are of particular interest. 【0163】 The method may further include administering one or more additional agents for treating cancer, such as one or more cytokine fusion proteins described herein, chemotherapeutic agents (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncological agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2, VEGF inhibitors), cell therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses, radiotherapy, etc. 【0164】 In embodiments, the fusion proteins described herein may be administered together with one or more inducible additional cytokine fusion proteins. For example, a fusion protein containing an IL-2 polypeptide, as described herein, may be administered together with a fusion protein containing an IL-12 polypeptide, an IFN polypeptide, a different IL-2 polypeptide, or a combination thereof. A fusion protein containing an IL-12 polypeptide, as described herein, may be administered together with a fusion protein containing an IL-2 polypeptide, an IFN polypeptide, a different IL-12 polypeptide, or a combination thereof. A fusion protein containing an IFN polypeptide, as described herein, may be administered together with a fusion protein containing an IL-2 polypeptide, an IL-12 polypeptide, a different IFN polypeptide, or a combination thereof. 【0165】 In some preferred embodiments, a first fusion protein containing any one amino acid sequence of SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, and 636-646 may be administered together with a second different fusion protein containing any one amino acid sequence of SEQ ID NOs: 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578. In some preferred embodiments, a first fusion protein containing any one amino acid sequence of 368-371, 434-440, 453-519, or 523-538 may be administered together with a second different fusion protein containing any one amino acid sequence of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 421-430, and 539-578. In some preferred embodiments, a first fusion protein containing any one amino acid sequence of SEQ ID NOs. 421-430 and 539-578 may be administered together with a second different fusion protein containing any one amino acid sequence of SEQ ID NOs. 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538 or any combination thereof. 【0166】 Further exemplary agents that may be administered in combination with one or more inducible cytokine fusion proteins described herein include, but are not limited to, cytokines (e.g., IL-2, IL-15), signal-inducing inhibitors (e.g., BRAF inhibitors or MEK inhibitors), checkpoint inhibitors (e.g., PDL-1, PD-1, CTLA-4), c-met inhibitors, kinase inhibitors (e.g., VGEF inhibitors), proteasome inhibitors, mTOR inhibitors, and angiogenesis inhibitors. 【0167】 Preferred immuno-oncological agents are anti-PD-L1 agents or anti-PD-1 agents. Exemplary PD-1 and / or PD-L1 inhibitors include, but are not limited to, spartalizumab, camrelizumab, cintilimab, tislerizumab, tripalimab, dostallimab, INCMGA00012, AMP-224, and AMP-514. In embodiments, the fusion proteins disclosed as 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646, 368-371, 434-440, 453-519, 523-538, 421-430, and 539-578 may be administered in combination with checkpoint inhibitors, such as PDL-1, PD-1, or CTL-4. 【0168】 This specification discloses pharmaceutical formulations or compositions containing a chimeric polypeptide and a pharmaceutically acceptable carrier. The compositions provided herein are suitable for in vitro or in vivo administration. A pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable, that is, the material is administered to a subject without causing undesirable biological effects or interacting with other components of the pharmaceutical formulation or composition containing it in a harmful manner. The carrier is selected to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject. 【0169】 Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 stThis is described in Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to make it isotonic, although the formulation may be hypertonic or hypotonic if desired. Examples of pharmaceutically acceptable carriers include, but are not limited to, sterile water, physiological saline, buffers such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release formulations, such as semipermeable matrices of solid hydrophobic polymers containing immunogenic polypeptides. The matrix may be in the form of a molded article, such as a membrane, liposomes, or microparticles. Certain carriers may be more preferred depending on, for example, the route of administration and the concentration of the composition to be administered. The carrier is suitable for administering chimeric polypeptides or nucleic acid sequences encoding chimeric polypeptides to humans or other subjects. 【0170】 Pharmaceutical preparations or compositions are administered in multiple ways, depending on whether topical or systemic treatment is desired and the area being treated. Compositions are administered by any of several forms of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, spray / inhalation, or via bronchoscopy. In some embodiments, compositions are administered topically (non-systemically), including into tumors, joints, subarachnoid spaces, etc. 【0171】 Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including physiological saline and buffered media. Parenteral vehicles include sodium chloride solution, dextrose-Ringer's solution, dextrose and sodium chloride, lactated Ringer's solution, or fixative oils. Intravenous vehicles include fluid and nutrient supplements, electrolyte supplements (e.g., dextrose-Ringer's solution-based). Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases and similar substances, are optionally present. 【0172】 Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powdered, or oily bases, thickeners, etc., are optionally necessary or desirable. 【0173】 Compositions for oral administration include powders or granules, suspensions or liquids made in water or a non-aqueous medium, capsules, pre-packaged preparations, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable. 【0174】 Selectively, chimeric polypeptides or nucleic acid sequences encoding chimeric polypeptides are administered by vectors. Multiple compositions and methods exist that can be used to deliver nucleic acid molecules and / or polypeptides to cells either in vitro or in vivo, for example, by expression vectors. These methods and compositions can generally be divided into two categories: viral delivery systems and non-viral delivery systems. Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide, or to therapeutically deliver nucleic acids. The components of the chimeric nucleic acids disclosed herein are typically linked in-frame to function as encoding a fusion protein. 【0175】 As used herein, a plasmid or viral vector is an active agent that transports the nucleic acid of this disclosure into a cell without degradation and includes a promoter that results in the expression of the nucleic acid molecule and / or polypeptide in the cell to which it was delivered. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindbis and other RNA viruses, such as those having the HIV skeleton. Any viral family that shares their properties that make these viruses suitable for use as vectors is also preferred. Retroviral vectors are described in general in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated herein by reference with respect to vectors and methods for making them. The construction of replication-deficient adenoviruses has been described (Berkner et al., J.Virol.61:1213-20 (1987), Massie et al., Mol. Cell.Biol.6:2872-83 (1986), Haj-Ahmad et al., J.Virol.57:267-74 (1986), Davidson et al., J.Virol.61:1226-39 (1987), Zhang et al., BioTechniques 15:868-72 (1993)). The advantage and use of these viruses as vectors is that their ability to spread to other cell types is limited because, although they can replicate in the initial infected cell, they cannot form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and several other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors. 【0176】 The polypeptides and / or nucleic acid molecules provided may be delivered by virus-like particles. Virus-like particles (VLPs) consist of viral proteins (or more) derived from the structural proteins of a virus. Methods for producing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004). 【0177】 The polypeptides provided can be delivered by subviral dense bodies (DBs). DBs transport proteins into target cells by membrane fusion. Methods for constructing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003). 【0178】 The polypeptides to be provided may be delivered by tegument aggregates. Methods for preparing and using tegument aggregates are described in International Publication No. WO2006 / 110728. 【0179】 Nonviral delivery methods may include expression vectors containing nucleic acid sequences encoding nucleic acid molecules and polypeptides, where the nucleic acids are functionally linked to expression regulatory sequences. Suitable vector skeletons include, for example, those conventionally used in the art, such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. These regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein-binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, stop sequences, polyadenylation sequences, and introns. Such vectors may also be used to create chimeric polypeptides by expression in suitable host cells, such as CHO cells. 【0180】 Preferred promoters that control transcription from vectors in mammalian host cells can be obtained from a variety of sources, e.g., from the genomes of viruses such as polyomas, monkey virus 40 (SV40), adenoviruses, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV); or from heterologous mammalian promoters, e.g., the β-actin promoter or the EF1α promoter; or from hybrid or chimeric promoters (e.g., a CMV promoter fused to a β-actin promoter). Of course, promoters from host cells or related species are also useful herein. 【0181】 Enhancers generally refer to DNA sequences that function at an indeterminate distance from the transcription start site, and can be located either at the 5' or 3' end of the transcription unit. Furthermore, enhancers can be found within introns or within the coding sequence itself. They are typically 10–300 base pairs (bp) in length and function in cis. Enhancers generally function to enhance transcription from nearby promoters. Enhancers may also contain response elements that mediate the regulation of transcription. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), but it is typical to use enhancers from eukaryotic viruses for normal expression. Preferred examples include the SV40 enhancer located behind the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located behind the origin of replication, and the adenovirus enhancer. 【0182】 Promoter and / or enhancer regions may be induceable (e.g., chemically or physically modulated). Chemically modulated promoters and / or enhancers may be modulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically modulated promoters and / or enhancers may be modulated, for example, by environmental factors, such as temperature and light. Optionally, promoter and / or enhancer regions may act as constitutive promoters and / or enhancers to maximize the expression of the region of the transcription unit being transcribed. In some vectors, promoter and / or enhancer regions may be activated in a cell-type specific manner. Optionally, in some vectors, promoter and / or enhancer regions may be activated in all eukaryotic cells, regardless of cell type. Preferred promoters of this type include the CMV promoter, SV40 promoter, β-actin promoter, EF1α promoter, and retroviral long-chain terminal repeats (LTRs). 【0183】 Vectors may also contain, for example, origins of replication and / or markers. Marker genes can confer a selectable phenotype to cells, such as antibiotic resistance. Marker products are used to determine whether the vector has been delivered to the cells and, once delivered, whether it is being expressed. Examples of selective markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidine. Such selective markers are successfully transferred into mammalian host cells, and the transformed mammalian host cells can survive under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors may contain tag sequences designed to facilitate the handling or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG® tag (Kodak, New Haven, Conn.) sequences, are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere in the polypeptide, for example, at either the carboxyl or amino terminus. 【0184】 As used herein, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by peptide bonds. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to suggest a particular size or number of amino acids constituting a molecule, and that the peptides of the present invention may contain a few or fewer amino acid residues. Subjects, as used throughout, may be vertebrates, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animals. The terms do not imply a particular age or sex. Therefore, adult and neonatal subjects are intended to encompass both males and females. As used herein, patient or subject may be used interchangeably and may refer to a subject having a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects. 【0185】 Individuals at risk of developing a disease or disability may have a genetic predisposition to the disease or disability, for example, they may have a family history, have mutations in their genes that cause the disease or disability, or may show early signs or symptoms of the disease or disability. Individuals currently having a disease or disability may have one or more symptoms of the disease or disability and may have been diagnosed with the disease or disability in the past. 【0186】 The methods and agents described herein are useful in both prophylactic and therapeutic treatments. For prophylactic use, a therapeutically effective amount of the chimeric polypeptide or chimeric nucleic acid sequence encoding a chimeric polypeptide described herein is administered before the onset of disease (e.g., before the appearance of obvious signs of cancer or inflammation) or during the initial stages of disease (e.g., when the initial signs and symptoms of cancer or inflammation appear). Prophylactic administration may occur over several days to several years before the manifestation of symptoms of cancer or inflammation. Prophylactic administration may be used, for example, in the prophylactic treatment of subjects diagnosed as genetically predisposed to cancer. Therapeutic treatments include administering a therapeutically effective amount of the chimeric polypeptide or chimeric nucleic acid sequence encoding a chimeric polypeptide described herein after the diagnosis or progression of cancer or inflammation (e.g., autoimmune disease). Prophylactic use may also be applied when a patient is receiving treatment in which inflammation is anticipated, such as chemotherapy. 【0187】 According to the methods taught herein, subjects are administered an effective dose of a drug (e.g., a chimeric polypeptide). The terms effective dose and effective dosage are used interchangeably. The term effective dose is defined as any amount required to produce a desired physiological response. The effective dose and the plan for administering the drug may be determined experimentally, and making such determinations is within the scope of the art. The dosage range for administration should be large enough to produce the desired effect, in which one or more symptoms of a disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so high as to cause significant adverse side effects, such as unwanted cross-reactivity or anaphylactic reactions. Typically, the dosage varies depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or the presence or absence of other drugs included in the treatment plan, and can be determined by those skilled in the art. The dosage may be adjusted by individual physicians if there are any contraindications. The dosage may vary and may be administered in doses once or more times daily over a day or several days. Guidelines for appropriate dosages for a given class of pharmaceutical products can be found in the literature. 【0188】 As used herein, the terms treatment, cure, or treat mean a disease or condition, or a method of reducing the effects of the symptoms of a disease or condition. Therefore, in the methods of this disclosure, treatment may mean a reduction of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in the severity of an existing disease or condition, or the symptoms of a disease or condition. For example, a method of treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Therefore, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the original level or control level. It is understood that treatment does not necessarily mean a cure or complete elimination of a disease, condition, or the symptoms of a disease or condition. 【0189】 As used herein, the terms to prevent, prevent, and inhibit a disease or disorder mean an action that occurs before or at approximately the time a subject begins to exhibit one or more symptoms of a disease or disorder, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, which suppresses or delays the onset or exacerbation of one or more symptoms of the disease or disorder. As used herein, references to reducing, mitigating, or inhibiting include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level. Such terms may, but are not necessarily, include complete elimination. 【0190】 IL-2 variants that are selective for IL2Rαβγ when compared to IL2Rβγ have been developed (Shanafelt, AB, et al., 2000, Nat Biotechnol. 18:1197-202, Cassell, DJ, et al., 2002, Curr Pharm Des., 8:2171-83). These variants have amino acid substitutions that reduce their affinity for IL2RB. Since IL-2 has an undetectable affinity for IL2RG, these variants consequently have a reduced affinity for the IL2Rβγ receptor complex and a reduced ability to activate IL2Rβγ-expressing cells, but they retain their ability to bind to IL2RA and to bind to and activate the IL2Rαβγ receptor complex. 【0191】 One of these variants, IL2 / N88R (Bay 50-4798), was clinically tested as a less toxic form of...

Claims

[Claim 1] A conditionally active IL-2 fusion protein comprising a fusion polypeptide covalently or noncovalently bound to a second polypeptide, The fusion polypeptide comprises a cytokine polypeptide [A], an inhibitory moiety [D], a half-life extension moiety [H], and a protease-cleavable polypeptide linker; The second polypeptide and the inhibitory portion of the fusion polypeptide are complementary and together form a functional binding site that is a cytokine-specific antibody Fab fragment; The cytokine polypeptide is an IL-2 polypeptide; The inhibiting portion is VH-CH1 and the second polypeptide contains complementary VL-CL, or the inhibiting portion is VL-CL and the second polypeptide contains complementary VH-CH1; The portion that extends the half-life is human serum albumin or an antigen-binding polypeptide that binds to human serum albumin; The cytokine polypeptide, the inhibitory portion, and the half-life extension portion are functionally linked by the protease-cleavable polypeptide linker, the fusion polypeptide has a weakened IL-2 receptor activating effect, and the IL-2 receptor activating effect of the fusion polypeptide is about one-tenth or less of the IL-2 receptor activating effect of a polypeptide containing an IL-2 polypeptide generated by cleavage of the protease-cleavable polypeptide linker. The fusion polypeptide has the formula: [A]-[L1]-[H]-[L2]-[D], where [L1] is a protease-cleavable polypeptide linker and [L2] is an optionally protease-cleavable polypeptide linker. An IL-2 fusion protein that becomes active under the aforementioned conditions. [Claim 2] The conditionally active IL-2 fusion protein according to claim 1, wherein the fusion polypeptide comprises VH-CH1 and the second polypeptide comprises VL-CL. [Claim 3] The conditionally active IL-2 fusion protein according to claim 1, wherein the fusion polypeptide comprises VL-CL and the second polypeptide comprises VH-CH1. [Claim 4] A pharmaceutical composition comprising a conditionally active IL-2 fusion protein according to any one of claims 1 to 3, for use in the treatment of cancer or a cancer-related viral infection, wherein the use comprises administering the pharmaceutical composition to a subject requiring treatment for cancer or a cancer-related viral infection. [Claim 5] A nucleic acid encoding a conditionally active IL-2 fusion protein as described in any one of claims 1 to 4. [Claim 6] A vector comprising the nucleic acid described in claim 5. [Claim 7] A host cell containing the vector according to claim 6.

Images