Activatable cytokine polypeptides and methods of use thereof

Abstract

To provide an activatable cytokine polypeptides and methods of use thereof.SOLUTION: The present disclosure features fusion proteins that are conditionally active variants of a cytokine of interest. In one aspect, full-length polypeptides of the invention have reduced or minimal cytokine-receptor activating activity even though they contain a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g., a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling. Typically, the fusion proteins further comprise an in vivo half-life extension element, which may be cleaved from the cytokine in a tumor microenvironment.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 62 / 935,605, filed November 14, 2019, the entire teachings of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on November 12, 2020, is named 761146_000146_SL.txt, and is 2,974,211 bytes in size. [Background technology]

[0003] The development of mature immunocompetent lymphoid cells from less committed precursors, their subsequent antigen-induced immune responses, and the suppression of these unwanted autoreactive responses are highly 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 the development of Treg cells in the thymus and critically regulates several essential aspects of mature peripheral Tregs and conventional antigen-activated T cells. IL-2 has been extensively studied in part due to its potent T cell growth factor activity in vitro, which has presented a potential means for directly enhancing immunity, e.g., in cancer and AIDS-HIV patients, or as a target for combating unwanted responses, e.g., graft rejection and autoimmune diseases. Although in vitro studies of IL-2 have provided a compelling rationale for these studies, the function of IL-2 in vivo appears to be much more complex, as first demonstrated in IL-2-deficient mice (Sadlack et al., 1993, 1995), which exhibited a rapidly fatal autoimmune syndrome rather than a loss of immunity. Later, similar observations were made when the genes encoding IL-2Rα (Il2ra) and IL-2Rβ (Il2rb) were individually ablated (Suzuki et al., 1995; Willerford et al., 1995).

[0004] The present invention relates to conditionally active and / or targeted cytokines for use in the treatment of cancer and other diseases dependent on immune up- or down-regulation. For example, the anti-tumor activity of several cytokines is well known and documented, and several cytokines are already being used therapeutically in humans. Cytokines such as interleukin-2 (IL-2) and interferon alpha (IFNα) have shown positive anti-tumor activity in patients with various tumors, such as metastatic renal carcinoma, hairy cell leukemia, Kaposi's sarcoma, melanoma, and multiple myeloma. Other cytokines, such as IFNβ, tumor necrosis factor (TNF) alpha, TNFβ, IL-1, IL-4, IL-6, IL-12, IL-15, and CSFs, have shown some anti-tumor activity against several types of tumors and are therefore the subject of further study. Summary of the Invention

[0005] Provided herein are therapeutic proteins, nucleic acids (e.g., DNA, RNA, mRNA) encoding such proteins, and methods and compositions using such proteins and nucleic acids for the treatment of diseases or disorders, such as proliferative diseases, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, etc. In one embodiment, the fusion protein has the amino acid sequence of any one of SEQ ID NOs: 193-271. Certain 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. The disclosure also relates to nucleic acids (e.g., DNA, RNA, mRNA) encoding the fusion proteins, methods for making the fusion proteins, compositions comprising the fusion proteins, and methods of using the fusion proteins to treat cancer, including combinations of two or more fusion proteins and one or more fusion proteins in combination with another therapeutic agent.

[0006] The present invention features fusion proteins that are conditionally active variants of a cytokine of interest. Cytokines of particular interest include IL-2, IL-12, and IFN. In one embodiment, the full-length polypeptides of the invention have reduced or minimal cytokine receptor activating activity despite containing a functional cytokine polypeptide. A cytokine, 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 a functional fragment or mutein or functional variant or subunit of any of the above, can bind to its receptor and confer signal transduction upon activation, e.g., by cleavage of a linker connecting the active cytokine to a blocking moiety, e.g., a steric blocking polypeptide. Optionally, the full-length polypeptide can include a blocking polypeptide moiety that also provides additional beneficial properties. For example, the full-length polypeptide may contain a blocking polypeptide portion that also extends serum half-life and / or directs the full-length polypeptide to a desired cytokine site of action. Alternatively, the full-length fusion polypeptide may contain a serum half-life-extending element and / or a targeting domain that are separate from the blocking polypeptide portion. Preferably, the fusion protein contains at least one element or domain that can extend circulating half-life in vivo. Preferably, this element is enzymatically removed at the desired location in the body (e.g., by protease cleavage in the tumor microenvironment), restoring the pharmacokinetic properties of the payload molecule (e.g., IL2, IL-12, IFNb, or IFNa) to substantially the same as those of the naturally occurring payload molecule. The fusion protein may be targeted to a desired cell or tissue.

[0007] The fusion polypeptide typically comprises a cytokine polypeptide [A], a blocking moiety [D], an optional half-life extending moiety [H], and a protease-cleavable polypeptide linker. The cytokine polypeptide, the blocking moiety, and, if present, the optional half-life extending moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated cytokine receptor activating activity, for example, the cytokine receptor activating activity of the fusion polypeptide is at least about 10-fold less than the cytokine receptor activating activity of a polypeptide containing the cytokine polypeptide generated by cleavage of the protease-cleavable linker. Some preferred fusion polypeptides have the formulas (I)-(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); wherein A is a cytokine polypeptide, D is a blocking moiety, H is a half-life extending moiety, L1 is a protease-cleavable polypeptide linker, L2 is a polypeptide linker that is optionally protease-cleavable, and L2' is a protease-cleavable polypeptide linker. L1 and L2, or L1 and L2', can 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 proteins may contain an IL-2 polypeptide. Fusion proteins containing an IL-2 polypeptide may comprise or consist of the 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. Fusion proteins disclosed as SEQ ID NOs: 257-300, 302-317, 325-353, 355-365, 366, 372-381, 383-385, 388-420, 579-608, 636-646 are also referred to herein as ACP289-ACP292, ACP296-ACP302, WW0301, ACP304-ACP306, ACP309-ACP313, WW0304, WW0306, WW0308, WW0309, WW0310, WW0311, WW0312, WW0313, WW0314, WW0315, WW0316, WW0317, WW0318, WW0319, WW0320, WW0321, WW0322, WW0323, WW0324, WW0325, WW0326, WW0327, WW0328, WW0329, WW0330, WW0331, WW0332, WW0333, WW0334, WW0335, WW0336, WW0337, WW0338, WW0339, WW0340, WW0341, WW0342, WW0343, WW0344, WW0345, WW0346, WW0348, WW0349, WW0350, WW0351, WW0352, WW0353, WW0354, WW0355, WW0356, WW035 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 comprise the amino acid sequence of SEQ ID NO: 272. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 286. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 362. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 336. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 348. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 363. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 580.

[0009] In embodiments, a fusion protein may contain IL-12. Fusion proteins containing an IL-12 polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 368-371, 434-440, 453-519, or 523-538. 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, a fusion protein may comprise the amino acid sequence of SEQ ID NO: 424. For example, a fusion protein may comprise the amino acid sequence of SEQ ID NO: 428. For example, a fusion protein may comprise the amino acid sequence of SEQ ID NO: 541. For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 556. For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 560. For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 568. For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 573.

[0010] In embodiments, the fusion protein contains IFN. Fusion proteins containing an IFN polypeptide may comprise or consist of the amino acid sequence of any one 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 can be covalently or non-covalently linked to a second polypeptide chain. For example, the fusion polypeptide can dimerize (i.e., form a dimer), or a portion of the fusion polypeptide can associate with another polypeptide, resulting in the formation of a functional binding site for, for example, a cytokine polypeptide or serum albumin. In certain embodiments, the second polypeptide chain and the blocking moiety on the fusion polypeptide are complementary and together form a functional binding site with specificity for the cytokine polypeptide contained in the fusion polypeptide. An exemplary functional binding site that can be formed by the blocking moiety of the fusion polypeptide and a complementary second polypeptide includes the antigen-binding site of an antibody, e.g., an Fab fragment or portion thereof. For example, one chain of the Fab that binds to the cytokine can be the blocking moiety of the fusion polypeptide, e.g., VH-CH1, and the complementary VL-CL can be part of the second polypeptide. In such cases, the blocking portion of the fusion protein, i.e., VH-CH1, and a second polypeptide comprising a complementary VL-CL can associate to form a functional binding site having specificity for the cytokine polypeptide (e.g., IL-2, IL-12, IFN alpha, IFN beta) contained in the fusion protein, thereby attenuating the cytokine polypeptide activity.

[0012] In embodiments, a fusion protein containing an IL-2 cytokine polypeptide may be covalently or noncovalently linked to a 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 NO: 263, 264, or 333. Such a second polypeptide may bind to a complementary VH-CH1 polypeptide contained in the fusion protein, for example, in SEQ ID NO: 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 embodiments, the fusion polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 362, 363, 325, 286, 579, 581 or 582, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263, 264 or 333. Fusion polypeptides disclosed as SEQ ID NOs: 362, 363, 325, 286, 579, 581, or 582 may be referred to as WW0520 (ACP378), WW0521 (ACP379), WW0548 (ACP406), WW0621 (ACP457), WW0729, WW0735, or WW0736, and 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). For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264.For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 286, and the second polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO:264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO:233. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:263. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:264. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:333. For example, the fusion protein may comprise or consist of SEQ ID NO:582, and the second polypeptide chain may comprise or consist of SEQ ID NO:263.For example, the fusion protein may comprise or consist of SEQ ID NO: 582 and the second polypeptide chain may comprise or consist of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of SEQ ID NO: 582 and the second polypeptide chain may comprise or consist of SEQ ID NO: 333.

[0014] As described herein, targeting can be achieved by the action of a blocking polypeptide moiety that also binds to the desired target, or by a targeting domain. A domain that recognizes a target antigen (e.g., a tumor-specific antigen) attached to a desired target can be linked to the cytokine via a cleavable or non-cleavable linker. When linked via a non-cleavable linker, the targeting domain can also serve to retain the cytokine within the tumor, and can be considered a retention domain. The targeting domain does not necessarily need to be directly linked to the payload molecule; it can also be directly linked to another element of the fusion protein. This is particularly true when the targeting domain is linked via a cleavable linker.

[0015] In one aspect, a fusion polypeptide is provided that includes a cytokine polypeptide, or a functional fragment, mutein, functional variant, or subunit thereof, and a blocking moiety, such as a steric blocking domain. The blocking 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 or linker, or within the blocking moiety. For example, if the cytokine polypeptide is fused to the blocking moiety via a linker containing a protease cleavage site, the cytokine polypeptide can be released from the blocking moiety and bind to its receptor via protease-mediated cleavage of the linker. The linker is designed to be cleaved at the desired site of cytokine action, for example, in the tumor microenvironment, while avoiding off-target cytokine action and reducing the overall toxicity of cytokine therapy.

[0016] The blocking moiety may also function as a serum half-life extending element. In some embodiments, the fusion polypeptide further comprises a separate serum half-life extending element. In some embodiments, the fusion polypeptide further comprises a targeting domain. In various embodiments, the serum half-life extending element is a water-soluble polypeptide, e.g., polyethylene glycol (PEG), optionally branched or multiarmed, full-length human serum albumin (HSA) or a fragment that maintains binding to FcRn, an Fc fragment, or a nanobody that binds directly to FcRn or to human serum albumin.

[0017] In addition to the serum half-life extending element, the pharmaceutical compositions described herein preferably include at least one or more targeting domains that bind to one or more target antigens or to one or more regions on a single target antigen. It is contemplated herein that the polypeptide constructs of the present invention are cleaved at a protease cleavage site, for example, in a disease-specific microenvironment or in the subject's blood, and that the targeting domain(s) bind to the target antigen on the target cell. The 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 diseases, neoplastic diseases, inflammatory diseases, immunological disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, or host-versus-graft disease.

[0018] In some embodiments, the target antigen is a cell surface molecule, e.g., a protein, lipid, or polysaccharide. In some embodiments, the target antigen is on a tumor cell, a virus-infected cell, a bacteria-infected cell, a damaged red blood cell, an arterial plaque cell, or a fibrous tissue cell.

[0019] In some cases, the target antigen is 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 activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 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 containing 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 targeting polypeptides independently comprise 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 targeting polypeptide specifically binds to a cell surface molecule. In some embodiments, the targeting polypeptide specifically binds to a tumor antigen. In some embodiments, the targeting 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 targeting 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 targeting polypeptide serves as a retention domain and is attached to the cytokine via a non-cleavable linker.

[0021] As described herein, a cytokine blocking moiety can bind to a cytokine and thereby block activation of the cytokine's cognate receptor.

[0022] The present disclosure also relates to nucleic acids, eg, DNA, RNA, mRNA, that encode the conditionally active proteins described herein, as well as vectors and host cells containing such nucleic acids.

[0023] The present disclosure also relates to pharmaceutical compositions containing the conditionally active proteins, nucleic acids encoding the conditionally active proteins, and vectors and host cells containing such nucleic acids. Typically, pharmaceutical compositions contain one or more physiologically acceptable carriers and / or excipients.

[0024] The present disclosure also relates to methods of treatment comprising administering an effective amount of a conditionally active protein, a nucleic acid encoding the conditionally active protein, a vector or host cell containing such a nucleic acid, or any of the above pharmaceutical compositions to a subject in need thereof. Typically, the subject has or is at risk of developing a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease, or host-versus-graft disease.

[0025] The present disclosure also relates to the use of conditionally active proteins, nucleic acids encoding the conditionally active proteins, vectors or host cells containing such nucleic acids, and pharmaceutical compositions of any of the above, to treat a subject in need thereof. Typically, the subject has or is at risk of developing a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease, or host-versus-graft disease.

[0026] The present disclosure also relates to the use of a conditionally active protein, a nucleic acid encoding a conditionally active protein, a vector or a host cell containing such a nucleic acid, for the manufacture of a medicament for treating a disease, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease or host-versus-graft disease. [Brief explanation of the drawings]

[0027] [Figure 1A] Figures 1A-1E are a series of graphs showing the activity of IL-2 fusion proteins in a HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of the control IL-2. EC50 values ​​for each IL-12 are shown in the table. Figures 1A-1E also show the results of a protein cleavage assay for each IL-2 polypeptide. 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 a HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of the control IL-2. EC50 values ​​for each IL-12 are shown in the table. Figures 1A-1E also show the results of a protein cleavage assay for each IL-2 polypeptide. 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 a HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of the control IL-2. EC50 values ​​for each IL-12 are shown in the table. Figures 1A-1E also show the results of a protein cleavage assay for each IL-2 polypeptide. 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 a HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of the control IL-2. EC50 values ​​for each IL-12 are shown in the table. Figures 1A-1E also show the results of a protein cleavage assay for each IL-2 polypeptide. 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 a HEKBlue IL-2 reporter assay in the presence of HSA. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of the control IL-2. EC50 values ​​for each IL-12 are shown in the table. Figures 1A-1E also show the results of a protein cleavage assay for each IL-2 polypeptide. 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]1 is a graph showing the activity of the non-cleavable control WW0729 / 523, also showing the results of a protein cleavage assay for the non-cleavable control. [Figure 2A] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2B] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2C]2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2D] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2E]2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2F] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2G]2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2H] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2I]2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2J] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2K]2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 2L] 2A-2L are a series of graphs showing the activity of fusion proteins in an IL-2 luciferase reporter assay. Filled squares represent the activity of uncleaved IL-2 polypeptide (intact), and open squares represent the activity of cleaved IL-2 polypeptide (truncated). Circles represent the activity of the control (human IL-2). The EC50 value for each is shown in the table (human IL-2). The EC50 value for each is shown in the table. Test constructs 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), WW0048 / WW0524 (Figure 2L). [Figure 3A]Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3B] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3C] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3D]Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3E] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3F] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3G]Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3H] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3I] Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), WW0520 / 524 (Figure 3J). [Figure 3J]Figures 3A-3J are a series of graphs showing the activity of fusion proteins in an IL-2 T-Blast assay. Squares represent the activity of the uncleaved IL-2 polypeptide (intact), and triangles represent the activity of the cleaved polypeptide (truncated). Circles represent the activity of control human IL-2. EC50 values ​​for each are shown in the table. 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 3I), 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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [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 (mean + / - standard error) over time for mice treated with the indicated doses of each fusion protein. The data demonstrate a dose-dependent decrease in tumor volume over time. [Figure 5A] Figures 5A-5C are a series of spider graphs showing the activity of fusion proteins in the MC38 mouse syngeneic model, corresponding to the data shown in Figures 4A-4G. Each line in the graph represents the tumor volume over time for a single mouse. Figure 5A includes data corresponding to vehicle treatment and the fusion proteins WW0517 and WW0520 / 523. [Figure 5B] Figures 5A-5C are a series of spider graphs showing the activity of fusion proteins in the MC38 mouse syngeneic model, corresponding to the data shown in Figures 4A-4G. Each line in the graph represents the tumor volume over time for a single mouse. Figure 5B contains data corresponding to treatment with fusion proteins WW0548 / 0524, WW0548 / 0556, and WW0475. [Figure 5C] Figures 5A-5C are a series of spider graphs showing the activity of fusion proteins in the MC38 mouse syngeneic model, corresponding to the data shown in Figures 4A-4G. Each line in the graph represents the tumor volume over time for a single mouse. Figure 5C contains data corresponding to treatment with fusion proteins WW0619 and WW0621 / 0523. [Figure 6A] Figures 6A-6C are a series of spider graphs showing the effect of fusion proteins on body weight in the 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 the fusion proteins WW0517 and WW0520 / 523. [Figure 6B]Figures 6A-6C are a series of spider graphs showing the effect of fusion proteins on body weight in the 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 contains data corresponding to treatment with fusion proteins WW0548 / 0524, WW0548 / 0556, and WW0475. [Figure 6C] Figures 6A-6C are a series of spider graphs showing the effect of fusion proteins on body weight in the 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 6C contains 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 a HEKBlue IL-12 reporter assay. Figures 7A-7Q show IL-12 / STAT4 activation compared between human p40 / mouse p35 IL-12 or human IL-12 fusion proteins and chimeric IL-12 (mouse p35 / human p40) or recombinant human IL-12 (control). Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of the control. 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 an IL12 T-Blast assay. Activation of IL-12 signaling is shown for human p40 / mouse p35 IL12 or human IL12 fusion proteins compared with control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of control chimeric IL-12 or recombinant human IL-12. 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 an IL12 T-Blast assay. Activation of IL-12 signaling is shown for human p40 / mouse p35 IL12 or human IL12 fusion proteins compared with control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of control chimeric IL-12 or recombinant human IL-12. 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 an IL12 T-Blast assay. Activation of IL-12 signaling is shown for human p40 / mouse p35 IL12 or human IL12 fusion proteins compared with control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of control chimeric IL-12 or recombinant human IL-12. 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 an IL12 T-Blast assay. Activation of IL-12 signaling is shown for human p40 / mouse p35 IL12 or human IL12 fusion proteins compared with control, chimeric IL-12 (human p40 / mouse p35 IL12), or recombinant human IL12. Squares represent the activity of the uncleaved IL-12 polypeptide (intact), and triangles represent the activity of the cleaved IL-12 polypeptide (truncated). Circles represent the activity of control chimeric IL-12 or recombinant human IL-12. 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. Panels A-B show the activation of IL-12 signaling compared between human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). Panel C shows the activation of IL-12 signaling compared between human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Filled squares represent the activity of the uncleaved IL-12 polypeptide (intact), and open squares represent the activity of the cleaved polypeptide (truncated). 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. Panels A-B show the activation of IL-12 signaling compared between human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). Panel C shows the activation of IL-12 signaling compared between human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Filled squares represent the activity of the uncleaved IL-12 polypeptide (intact), and open squares represent the activity of the cleaved polypeptide (truncated). 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. Panels A-B show the activation of IL-12 signaling compared between human p40 / mouse p35 IL-12 fusion protein and recombinant human IL-12 (control). Panel C shows the activation of IL-12 signaling compared between human IL-12 (human p40 / human p35 IL-12) fusion protein and recombinant human IL-12. Filled squares represent the activity of the uncleaved IL-12 polypeptide (intact), and open squares represent the activity of the cleaved polypeptide (truncated). 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 a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10B] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10C] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10D] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10E]Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10F] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10G]Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10H] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10I]Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 10J] Figures 10A-10J are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. Figures 10A-10J show activation of the IFN-α / β pathway compared between mouse IFNα fusion proteins and mouse IFNα (control). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (mouse IFNα). EC50 values ​​for each are shown in the table. Figures 10A-10J also show the results of a protein cleavage assay for each IFNα fusion protein. Both the cleaved and uncleaved forms of each IFNα fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 11A] 11A-11B are graphs showing results from a HEK-Blue IL-12 reporter assay performed on a human p40 / mouse p35 IL-12 fusion protein before and after protease cleavage. Constructs ACP35 (A) and ACP34 (B) were tested. The assay was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirm that the IL-12 protein fusion protein is active. [Figure 11B]11A-11B are graphs showing results from a HEK-Blue IL-12 reporter assay performed on a human p40 / mouse p35 IL-12 fusion protein before and after protease cleavage. Constructs ACP35 (A) and ACP34 (B) were tested. The assay was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirm that the IL-12 protein fusion protein is active. [Figure 12A] Figures 12A-12F show a series of graphs depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that the truncated IL-12 is more active than the intact 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 a protein cleavage assay are shown. Both the cleaved and uncleaved forms of the fusion protein ACP11 were run on an SDS-PAGE gel. As can be seen in the gel, cleavage was complete. [Figure 14] 1 is a schematic diagram depicting a non-limiting example of an inducible cytokine protein, where the construct is activated by protease cleavage of the linker connecting the 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 confirm that the IL12 protein fusion proteins are active. 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 confirm that the IL12 protein fusion proteins are active. 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 confirm that the IL12 protein fusion proteins are active. 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 confirm that the IL12 protein fusion proteins are active. Each proliferation assay was performed with or without HSA. [Figure 16A] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16B] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16C] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16D] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16E] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 16F] 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 a WEHI279 cell viability assay. Each assay was performed using medium containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17A] 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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17B]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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17C] 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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17D] 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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17E] 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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 17F]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 a B16 reporter assay. Each assay was performed using medium containing (+HSA) or not containing (-HSA). Each fusion protein contained an anti-HSA binder, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 18A] 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 can be seen in the gel, cleavage was complete. [Figure 18B] 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 can be seen in the gel, cleavage was complete. [Figure 19A] 19A-19B are a series of graphs (A and B) showing the activity of exemplary IFNγ fusion proteins before and after protease cleavage using a B16 reporter assay. Each assay was performed using culture medium containing HSA, and each fusion protein contains an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 19B] 19A-19B are a series of graphs (A and B) showing the activity of exemplary IFNγ fusion proteins before and after protease cleavage using a B16 reporter assay. Each assay was performed using culture medium containing HSA, and each fusion protein contains an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 20A]20A-20B are a series of graphs (A and B) showing the activity of exemplary IFNα fusion proteins before and after cleavage using a B16 reporter assay. Each assay was performed using medium containing HSA, and each fusion protein contains an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 20B] 20A-20B are a series of graphs (A and B) showing the activity of exemplary IFNα fusion proteins before and after cleavage using a B16 reporter assay. Each assay was performed using medium containing HSA, and each fusion protein contains an anti-HSA binder. Both the uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 21A] Figures 21A-21D are a series of graphs showing the results of a tumor growth study using the MC38 cell line. Figures A-C show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (µg = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). Figure D shows the effect 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 a tumor growth study using the MC38 cell line. Figures A-C show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (µg = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). Figure D shows the effect 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 a tumor growth study using the MC38 cell line. Figures A-C show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (µg = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). Figure D shows the effect 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 a tumor growth study using the MC38 cell line. Figures A-C show the effect of IFNγ and IFNγ fusion proteins on tumor growth when injected intraperitoneally (IP) using different dose levels and schedules (µg = micrograms, BID = twice daily, BIW = twice weekly, QW = weekly). Figure D shows the effect of intratumoral (IT) injection of IFNγ and IL-2 on tumor growth. [Figure 22A] 22A-22B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP51 (A) and ACP52 (B)) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains an anti-HSA binder and a tumor tropism domain. [Figure 22B] 22A-22B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP51 (A) and ACP52 (B)) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains an anti-HSA binder and a tumor tropism domain. [Figure 23A] 23A-23B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains IFNγ fused directly to albumin. [Figure 23B] 23A-23B are a series of graphs showing the activity of exemplary IFNγ fusion proteins (ACP53 and ACP54) cleaved by MMP9 protease compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains IFNγ fused directly to albumin. [Figure 24A]Figures 24A-24D are graphs showing 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 based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24A shows results for the IL-2 constructs ACP132 and ACP133 with and without albumin. [Figure 24B] Figures 24A-24D are graphs showing 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 based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24B shows results for truncated and uncleaved forms of the IL-2 construct ACP16. Results of a proteolytic cleavage assay for the truncated and uncleaved forms of ACP16 are also shown. [Figure 24C] Figures 24A-24D are graphs showing results from HEK-Blue IL-2 reporter assays performed on IL-2 fusion polypeptides and recombinant human IL-2 (Rec hIL-2). Analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24C shows results for cleaved and uncleaved forms of the IL-2 construct ACP153. Results from a protein cleavage assay are also shown. [Figure 24D] Figures 24A-24D are graphs showing 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 based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). Figure 24D shows results from a HEK-Blue IL-2 assay using wild-type cytokine, the intact fusion protein, and the protease-cleaved fusion protein. [Figure 25A]Figures 25A and 25B are two graphs showing the analysis of ACP16 (Figure 25A) and ACP124 (Figure 25B) in a 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 a 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 a 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 with serial dilutions of activatable hIL2 for 72 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable ACP16 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell activity was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay. Circles represent intact 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 a HEKBlue IL-12 reporter assay. Figure 26A shows 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 a HEKBlue IL-12 reporter assay. Figure 26B is a graph showing the analysis of ACP91 (a 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 a HEKBlue IL-12 reporter assay. Figure 26C is a graph showing the analysis of ACP136 (a 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. [Figure 27A] 27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27B] 27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27C] 27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27D] 27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27E] 27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 27C) are active in the B16-Blue IFN-α / β reporter assay. [Figure 27F]27A-27F are a series of graphs showing that the truncated murine IFNα1 polypeptides ACP31 (FIG. 27A), ACP125 (FIG. 27B), and ACP126 (FIG. 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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [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 a B16 IFNγ reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). [Figure 29A]Figures 29A-29B are two graphs showing the results of analyzing ACP31 (a murine IFNα1 fusion protein) and ACP11 (a human p40 / murine p35 IL12 fusion protein) in a tumor xenograft model. Figure 29A shows tumor volume over time in mice treated with 33 μg of ACP31 (circles), 110 μg of ACP31 (triangles), 330 μg of ACP31 (diamonds), and as controls, 1 μg of murine wild-type IFNα1 (dashed line, squares) and 10 μg of mIFNα1 (dashed line, small circles). Vehicle alone is represented by large open circles. The data show that tumor volume decreased over time in mice treated with ACP31 in a dose-dependent manner. [Figure 29B] Figures 29A-29B are two graphs showing the results of analyzing ACP31 (a murine IFNα1 fusion protein) and ACP11 (a human p40 / murine p35 IL12 fusion protein) in a tumor xenograft model. Figure 29B shows tumor volume over time in mice treated with 17.5 μg of ACP11 (squares), 175 μg of ACP31 (triangles), 525 μg of ACP31 (circles), and as controls, 2 μg of ACP04 (dashed line, triangles) and 10 μg of ACP04 (dashed line, diamonds). Vehicle alone is represented by a large open circle. The data show that tumor volume decreased over time in a dose-dependent manner in both ACP11- and ACP04-treated mice (human p40 / murine p35 IL12 fusion protein). [Figure 30A] Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 30B]Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 30C] Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 30D] Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 30E] Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 30F] Figures 30A-30F are a series of line graphs showing tumor volume over time in a mouse xenograft tumor model for 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). Each line represents a single mouse. [Figure 31A]Figures 31A-31C are three graphs showing the results of analyzing ACP16 and ACP124 in tumor xenograft models. Figure 31A shows tumor volume over time in mice treated with 4.4 μg of ACP16 (squares), 17 μg of ACP16 (triangles), 70 μg of ACP16 (inverted triangles), 232 μg of ACP16 (filled circles), and the control drugs 12 μg of wild-type IL-2 (dashed line, triangles) and 36 μg of wild-type IL-2 (dashed line, diamonds). Vehicle alone is represented by large open circles. The data show a dose-dependent decrease in tumor volume over time in mice treated with the higher concentrations 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 tumor volume over time in mice treated with 17 μg of ACP124 (squares), 70 μg of ACP124 (triangles), 230 μg of ACP124 (inverted triangles), and 700 μg of ACP124. Vehicle alone is represented by large open circles. [Figure 31C] Figures 31A-31C are three graphs showing the results of analyzing ACP16 and ACP124 in a tumor xenograft model. Figure 31C shows tumor volume over time in mice treated with 17 μg of ACP16 (triangles), 70 μg of ACP16 (circles), and 232 μg of ACP16 (filled circles), as well as control drugs 17 μg of ACP124 (dashed line, triangles), 70 μg of ACP124 (dashed line, diamonds), and 230 μg of ACP124 (dashed line, diamonds). Vehicle alone is represented by inverted black triangles. The data show that tumor volume decreased over time in a dose-dependent manner in mice treated with ACP16, but not 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] FIG. 33 is a graph showing tumor volume over time in a mouse xenograft model showing tumor growth in control mice (open circles) and AP16-treated mice (squares). [Figure 34A] Figures 34A-34D are a series of survival graphs showing survival over time of mice after treatment with cleavable fusion proteins. Figure 34A shows data for mice treated with vehicle alone (gray line), 17 μg ACP16 (black line), and 17 μg ACP124 (dashed line). [Figure 34B] Figures 34A-34D are a series of survival graphs showing survival over time of mice after treatment with cleavable fusion proteins. Figure 34B shows data for mice treated with vehicle alone (gray line), 70 μg ACP16 (black line), and 70 μg ACP124 (dashed line). [Figure 34C] Figures 34A-34D are a series of survival graphs showing survival over time of mice after treatment with cleavable fusion proteins. Figure 34C shows data for mice treated with vehicle alone (gray line), 232 μg ACP16 (black line), and 230 μg ACP124 (dashed line). [Figure 34D] Figures 34A-34D are a series of survival graphs showing survival over time of mice after treatment with cleavable fusion proteins. Figure 34D shows data for mice treated with vehicle alone (gray line), 232 μg ACP16 (black line), and 700 μg ACP124 (dashed line). [Figure 35] A series of line graphs showing the activity of fusion proteins in the MC38 mouse xenograft model. All groups of mice received a total of four doses, except for the highest three doses of APC132, for which lethal toxicity was detected after one week / two doses. Shown are vehicle alone (top), 17, 55, 70, and 230 μg of ACP16 (top row), 9, 28, 36, and 119 μg of ACP132 (middle row), and 13, 42, 54, and 177 μg of ACP21 (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 a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36B] Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36C]Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36D] Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36E]Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36F] Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36G]Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 36H] Figures 36A-36H are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 36A-36H show activation of the IFN-α / β pathway compared between human IFN-α fusion proteins and a control (human IFN-α). Squares represent the activity of the uncleaved IFN-α polypeptide (intact form), and triangles represent the activity of the cleaved IFN-α polypeptide (truncated form). Circles represent the activity of the control (human IFN-α). EC50 values ​​for each are shown in the table. The results confirm that the IFN-α fusion proteins are active and inducible. Figures 36A-36H also show the results of a protein cleavage assay for each IFN-α fusion protein. Both the cleaved and uncleaved forms of each IFN-α fusion protein were run on an SDS-PAGE gel. As can be seen on the gel, cleavage was complete. [Figure 37A]Figures 37A-37D are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 37A-37B show activation of the IFN-α / β pathway for mouse IFN-β fusion proteins compared to a control (mouse IFN-β). Figures 37C-37D show activation of the IFN-α / β pathway for human IFN-β fusion proteins compared to a control (human IFN-β). Squares represent the activity of the uncleaved IFN-β polypeptide (intact), and triangles represent the activity of the cleaved IFN-β polypeptide (truncated). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results confirm that the IFN-β fusion proteins are active and inducible. [Figure 37B] Figures 37A-37D are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 37A-37B show activation of the IFN-α / β pathway for mouse IFN-β fusion proteins compared to a control (mouse IFN-β). Figures 37C-37D show activation of the IFN-α / β pathway for human IFN-β fusion proteins compared to a control (human IFN-β). Squares represent the activity of the uncleaved IFN-β polypeptide (intact), and triangles represent the activity of the cleaved IFN-β polypeptide (truncated). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results confirm that the IFN-β fusion proteins are active and inducible. [Figure 37C] Figures 37A-37D are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 37A-37B show activation of the IFN-α / β pathway for mouse IFN-β fusion proteins compared to a control (mouse IFN-β). Figures 37C-37D show activation of the IFN-α / β pathway for human IFN-β fusion proteins compared to a control (human IFN-β). Squares represent the activity of the uncleaved IFN-β polypeptide (intact), and triangles represent the activity of the cleaved IFN-β polypeptide (truncated). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results confirm that the IFN-β fusion proteins are active and inducible. [Figure 37D]Figures 37A-37D are a series of graphs showing the activity of fusion proteins in a HEK-Blue IFN-α / β reporter assay. Figures 37A-37B show activation of the IFN-α / β pathway for mouse IFN-β fusion proteins compared to a control (mouse IFN-β). Figures 37C-37D show activation of the IFN-α / β pathway for human IFN-β fusion proteins compared to a control (human IFN-β). Squares represent the activity of the uncleaved IFN-β polypeptide (intact), and triangles represent the activity of the cleaved IFN-β polypeptide (truncated). Circles represent the activity of the control. The EC50 values ​​for each are shown in the table. The results confirm that the IFN-β fusion proteins are active and inducible. [Figure 38A] Figures 38A-38C are a series of graphs showing the activity of fusion proteins in a human PBMC assay. Figures A-C show activation of the IFNα pathway compared between human IFNα fusion proteins and a control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control human IFNα. EC50 values ​​for each are shown in the table. Analysis was based on quantitation of CXCL-10 (IP-10). The results confirm that the IFNα fusion proteins are active and inducible. [Figure 38B] Figures 38A-38C are a series of graphs showing the activity of fusion proteins in a human PBMC assay. Figures A-C show activation of the IFNα pathway compared between human IFNα fusion proteins and a control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control human IFNα. EC50 values ​​for each are shown in the table. Analysis was based on quantitation of CXCL-10 (IP-10). The results confirm that the IFNα fusion proteins are active and inducible. [Figure 38C]Figures 38A-38C are a series of graphs showing the activity of fusion proteins in a human PBMC assay. Figures A-C show activation of the IFNα pathway compared between human IFNα fusion proteins and a control (human IFNα). Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control human IFNα. EC50 values ​​for each are shown in the table. Analysis was based on quantitation of CXCL-10 (IP-10). The results confirm that the IFNα fusion proteins are 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 mean tumor volume over time for mice treated with 369 μg WW0610 (squares), 553 μg WW0610 (downward triangles), 830 μg WW0610 (upward triangles), and 1,245 μg WW0610 (circles). Vehicle alone is represented by filled circles. [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 for mice treated with 1,231 μg WW0815 (squares), 1,845 μg WW0815 (downward triangles), 2,770 μg WW0815 (upward triangles), and 4,154 μg WW0815 (circles). Vehicle alone is represented by filled circles. [Figure 39C] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39C shows the mean tumor volume over time for mice treated with 4.6 μg WW0644 (squares), 9.3 μg WW0644 (downward triangles), 19 μg WW0644 (upward triangles), and 37 μg WW0644 (circles). Vehicle alone is represented by filled circles. [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 for mice treated with 31 μg WW0816 (squares), 62 μg WW0816 (downward triangles), 123 μg WW0816 (upward triangles), and 247 μg WW0816 (circles). Vehicle alone is represented by filled circles. [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 WW0609 (squares), 830 μg WW0609 (downward triangles), and 1,320 μg WW0609 (upward triangles). [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 WW0610 (squares), 830 μg WW0610 (downward triangles), and 1,320 μg WW0610 (upward triangles). [Figure 39G] Figures 39A-39G show the results of analyzing INF fusion proteins in a syngeneic MC38 mouse tumor model. Figure 39G shows the mean tumor volume over time for mice treated with 0.3 μg WW0643 (squares), 1.5 μg WW0643 (downward triangles), 7.5 μg WW0643 (upward triangles), and 37.5 μg WW0643 (diamonds). Vehicle alone is represented by closed circles. [Figure 40A] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40B] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40C]Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40D] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40E] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40F] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 40G] Figures 40A-40G show a series of spider graphs showing the activity of the fusion protein in the MC38 xenograft model, corresponding to the data in Figures 39A-39G. Each line in the graph is the tumor volume over time for a single mouse. [Figure 41A] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41B] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41C] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41D] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41E] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41F] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 41G] Figures 41A-41G show the mean percent body weight over time for mice treated with the INF fusion proteins of Figures 39A-39G. [Figure 42A] Figures 42A-42E show the results of a B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after truncation. The relevant wild-type IFN was tested as a control. [Figure 42B] Figures 42A-42E show the results of a B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after truncation. The relevant wild-type IFN was tested as a control. [Figure 42C] Figures 42A-42E show the results of a B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after truncation. The relevant wild-type IFN was tested as a control. [Figure 42D] Figures 42A-42E show the results of a B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after truncation. The relevant wild-type IFN was tested as a control. [Figure 42E] Figures 42A-42E show the results of a B16 IFN reporter assay. The inducible interferon construct of interest was tested before and after truncation. The relevant wild-type IFN was tested as a control. [Figure 43] Binding data for ACP16, ACP10, and ACP11 are shown. [Figure 44A] Figures 44A-44D show the activity of cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244 and ACP247. [Figure 44B] Figures 44A-44D show the activity of cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244 and ACP247. [Figure 44C]Figures 44A-44D show the activity of cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244 and ACP247. [Figure 44D] Figures 44A-44D show the activity of cytokine fusion protein constructs ACP243, ACP244, ACP243, ACP244 and ACP247. [Figure 45] A series of spider graphs 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 the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54B] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54C] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54D] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54E]Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54F] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54G] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54H] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54I] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Fig. 54J] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54K] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54L] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54M] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the uncleaved protein and IL2 as a control. [Figure 54N] Figures 54A-54N show the results of a proliferation assay comparing the cleaved protein with the 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 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, and include IL-2 as a control. [Figure 56] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57A] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57B] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57C] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 57D] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 58] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59A] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59B] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59C] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59D] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59E] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59F] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59G] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59H] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59I] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59J] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59K] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59L] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59M] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59N] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59O] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59P]Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Q] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59R] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59S] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59T] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59U] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59V] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59W] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Z-1] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Y] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 59Z-2] Figures 56, 57A-57D, 58, 59A-59Z show the activity of cytokine fusion protein constructs. [Figure 60A] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60B]Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60C] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60D] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60E] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60F] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 60G] Figures 60A-60G show a series of spider graphs illustrating the effect of INF fusion proteins on body weight in an MC38 mouse xenograft model, corresponding to the data shown in Figures 41A-41G. Each line in the graph is the body weight of a single mouse over time. [Figure 61A]Figures 61A-B are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. A shows activation of the IFN-α / β pathway for construct WW0609 in the presence and absence of albumin. B shows activation of the IFN-α / β pathway for construct WW0643 in the presence and absence of albumin. Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (murine IFNα). The EC50 values ​​for each are shown in the table. [Figure 61B] Figures 61A-B are a series of graphs showing the activity of fusion proteins in a B16-Blue IFN-α / β reporter assay. A shows activation of the IFN-α / β pathway for construct WW0609 in the presence and absence of albumin. B shows activation of the IFN-α / β pathway for construct WW0643 in the presence and absence of albumin. Squares represent the activity of the uncleaved IFNα polypeptide (intact), and triangles represent the activity of the cleaved IFNα polypeptide (truncated). Circles represent the activity of the control (murine IFNα). The EC50 values ​​for each are shown in the table. DETAILED DESCRIPTION OF THE INVENTION

[0028] Disclosed herein are methods and compositions for designing and using constructs containing inducible cytokines. Cytokines are potent immune agonists, which is why they are considered promising therapeutic agents for oncology. However, cytokines have proven to have a very narrow therapeutic window. Cytokines have short serum half-lives and are also considered very potent. As a result, therapeutic administration of cytokines has resulted in undesirable systemic effects and toxicity. These are exacerbated by the need to administer large amounts of cytokine to achieve desired levels at the intended site of cytokine action (e.g., tumor). Unfortunately, due to cytokine biology and the inability to effectively direct and control their activity, cytokines have not achieved the desired clinical benefit in tumor treatment.

[0029] Disclosed herein are fusion proteins that overcome the challenges of toxicity and short half-life that have significantly limited the clinical use of cytokines in oncology. The fusion proteins contain a cytokine polypeptide with receptor agonist activity. However, in the context of the fusion protein, the cytokine receptor agonist activity is attenuated and the circulating half-life is extended. The fusion protein includes a protease cleavage site that is associated with the desired cytokine site of action (e.g., a 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 restricting cytokine action to the desired site of action, e.g., the tumor microenvironment. Protease cleavage at the desired site of action, e.g., the tumor microenvironment, liberates a form of cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein (typically at least about 100-fold more active than the fusion protein). The form of cytokine released upon cleavage of the fusion protein typically has a short half-life, often substantially similar to that of the naturally occurring cytokine, resulting in cytokine action being further localized to 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 directing of the active cytokine to the tumor microenvironment. The fusion proteins described herein, for the first time, enable the administration of effective therapeutic doses of cytokines to treat tumors, with cytokine action substantially limited to the tumor microenvironment and dramatically reducing and eliminating the cytokine's undesired systemic effects and toxicity.

[0030] The fusion proteins disclosed herein typically comprise a cytokine polypeptide [A], a blocking moiety [D], an optional half-life extending moiety [H], and a protease-cleavable polypeptide linker. The cytokine polypeptide, blocking moiety, and, if present, the optional half-life extending moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated cytokine receptor activating activity, for example, the cytokine receptor activating activity of the fusion polypeptide is at least about 10-fold less than the cytokine receptor activating activity of a polypeptide containing the cytokine polypeptide generated by cleavage of the protease-cleavable linker. Some preferred fusion polypeptides have the formulas (I)-(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); wherein [A] is a cytokine polypeptide, [D] is a blocking moiety, [H] is a half-life extending moiety, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, and [L2'] is a protease-cleavable polypeptide linker. [L1] and [L2], or [L1] and [L2'], can optionally have the same or different amino acid sequences and / or protease cleavage sites (if L2 is protease-cleavable).

[0031] The present disclosure further relates to pharmaceutical compositions containing the inducible fusion proteins and additional therapeutic agents, as well as nucleic acids encoding the polypeptides, and recombinant expression vectors and host cells for making such fusion proteins. Also provided herein are methods of using the fusion proteins of the present disclosure in the treatment of diseases, conditions, and disorders.

[0032] Unless otherwise specified, all terms of technical field, notation, and other scientific and technical terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms having commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a departure from what is commonly understood in the art. The techniques and procedures described or referred to herein are generally well understood and routinely employed using conventional methodologies by those of ordinary skill in the art, such as the widely used molecular cloning methodologies described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-specified protocols and conditions unless otherwise noted.

[0033] "Cytokine" is a well-known term of art that refers, inter alia, to any of a class of immunomodulatory proteins (e.g., interleukins or interferons) that are secreted by cells of the immune system and are regulators of the immune system. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include transforming growth factors, such as TGF-α and TGF-β (e.g., TGFbeta1, TGFbeta2, TGFbeta3); interferons, such as interferon-α, interferon-β, interferon-γ, interferon-kappa, and interferon-omega; interleukins, such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, , IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25; tumor necrosis factors, such as tumor necrosis factor alpha and lymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as fragments of such polypeptides (i.e., functional fragments as described above) that activate the cognate receptor for the cytokine. "Chemokine" is a term of art that refers to any of a family of small cytokines capable of inducing directional chemotaxis in nearby responding cells.

[0034] It is well known that cytokines have a short serum half-life, often only a few minutes or hours. Even cytokine forms with modified amino acid sequences intended to extend serum half-life but retain receptor agonist activity typically have short serum half-lives. As used herein, "short half-life cytokine" refers to a cytokine that circulates in the serum of a subject for a fairly short period of time, for example, 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 whose sequences have not been modified to provide a longer-than-normal half-life in the subject's body and to provide a polypeptide with a modified amino acid sequence intended to extend serum half-life but retain receptor agonist activity. This latter term does not imply the addition of a heterologous protein domain, such as a genuine half-life-extending element, such as serum albumin.

[0035] "Sortases" are transpeptidases that modify proteins by recognizing and cleaving carboxyl-terminal sorting signals embedded in or attached to the terminus of target proteins or peptides. Sortase A catalyzes the cleavage of an LPXTG motif (SEQ ID NO: 442) (where X is any standard amino acid) on target proteins between the Thr and Gly residues, which involves transient binding of the Thr residue to an active site Cys residue on the enzyme to form an enzyme-thioacyl intermediate. To complete the transpeptidation and create a peptide-monomer conjugate, a biomolecule bearing an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displacing sortase A and linking the two molecules.

[0036] As used herein, the term "steric blocker" refers to a polypeptide or polypeptide portion that may be covalently bound to a cytokine polypeptide, either directly or indirectly through other moieties such as a linker, e.g., in the form of a chimeric polypeptide (fusion protein), but is otherwise not covalently bound to the cytokine polypeptide. Steric blockers may bind to the cytokine polypeptide non-covalently, e.g., by electrostatic, hydrophobic, ionic, or hydrogen bonding. Steric blockers typically inhibit or block the activity of the cytokine moiety due to their proximity and relative size to the cytokine moiety. Steric blockers may also achieve blocking by employing large protein binding partners. One example of this is an antibody that binds to serum albumin; while the antibody itself may or may not be large enough to block activation or binding by itself, employing albumin provides sufficient steric blocking.

[0037] As used and described herein, a "half-life extending element" is a portion of a chimeric polypeptide that increases serum half-life and improves pK, for example, by altering its size (e.g., above the renal filtration cutoff), shape, hydrodynamic diameter, charge, or parameters of absorption, biodistribution, metabolism, and excretion.

[0038] As used herein, the terms "activatable," "activate," "induce," and "inducible" refer to the ability of a protein that is part of a fusion protein, i.e., a cytokine, to bind to its receptor and produce 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 a nucleic acid of the present disclosure into a cell without degradation and contains a promoter that drives expression of a cellular nucleic acid molecule and / or polypeptide in the cell to which it is delivered.

[0040] As used herein, the terms "peptide," "polypeptide," or "protein" are used broadly to refer to two or more amino acids linked by a peptide bond. 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 imply a particular size or number of amino acids comprising the molecule, and that the peptides of the present invention may contain fewer or more amino acid residues.

[0041] As used throughout, a "subject" may be a vertebrate, and more specifically, may be a mammal (e.g., a 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 newborn subjects, whether male or female, are intended to be encompassed.

[0042] As used herein, "patient" or "subject" may be used interchangeably and refer to a subject having a disease or disorder (e.g., cancer). The term patient or subject includes human and veterinary subjects.

[0043] As used herein, the terms "treatment," "treat," or "treating" refer to a method of reducing the effects of a disease or condition, or a symptom of a disease or condition. Thus, in the methods of the present disclosure, treatment can refer to reducing the severity of an existing disease or condition, or a symptom 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 completely. For example, a method of treating a disease is considered therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10 and 100% compared to the original or control level. It is understood that treatment does not necessarily mean a cure or complete elimination of the disease, condition, or symptoms of the disease or condition.

[0044] As used herein, the terms "prevent," "preventing," and "prevention" of a disease or disorder refer to an action that occurs before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder, such as administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, which inhibits or delays the onset or progression of one or more symptoms of the disease or disorder.

[0045] As used herein, references to "decrease," "reducing," or "inhibiting" 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 more, compared to a suitable control level. Such terms may, but do not necessarily, include complete elimination of a function or property, e.g., agonist action.

[0046] A "attenuated cytokine receptor agonist" is a cytokine receptor agonist that has reduced receptor agonist activity compared to a naturally occurring agonist for the cytokine receptor. A attenuated cytokine agonist may have agonist activity that is at most about 10-fold, at most about 50-fold, at most about 100-fold, at most about 250-fold, at most about 500-fold, at most about 1000-fold, or less than that of a naturally occurring agonist for the receptor. When a fusion protein containing a cytokine polypeptide described herein is described as being "attenuated" or having "attenuated activity," it is meant that the fusion protein is a attenuated cytokine receptor agonist.

[0047] An "intact fusion protein" is a fusion protein in which none of the domains have been removed, for example, by protease cleavage. Domains may be removable by protease cleavage or other enzymatic action, but this has not occurred when the fusion protein is "intact."

[0048] As used herein, a "moiety" refers to a portion of a molecule that has a distinct function within that molecule, and that function can be exerted by the moiety in the context of another molecule. A moiety can be a chemical entity with a specific function or a portion of a biological molecule with a specific function. For example, a "blocking moiety" 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 can be a protein domain, such as serum albumin. Blocking can be achieved by a steric blocker or a specific blocker. A steric blocker blocks by size and location rather than specific binding, and an example is serum albumin. A specific blocker blocks by specific interaction with the blocked moiety. Specific blockers must be tailored to fit a specific cytokine or activity domain, and steric blockers can be used regardless of the drug payload as long as they are large enough. If desired, the blocking moiety incorporated into the fusion proteins described herein can associate with another polypeptide to create a specific binding domain. For example, when a cytokine-binding fragment of an antibody is used as the blocking moiety, the fusion polypeptide can include a cytokine-specific scFv as the blocking moiety, or the fusion polypeptide can include a Fab half, e.g., VH-CH1 or VL-CL, as the blocking moiety, which can associate with a complementary chain, VL-CL or VH-CH1, respectively, to form a Fab fragment that specifically binds to the cytokine. As further described herein, the blocking moiety can block the activity of the fusion protein directly or when the blocking moiety is associated with another polypeptide, for example, when an anti-HSA scFv binds to HSA, or when a VH-CH1 polypeptide associates with a complementary VL-CL polypeptide and then 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 of some tumors and for its in vitro cytotoxic effects against various neoplastic lineages, but it was later proven to have strong pro-inflammatory effects that could endanger the human body in the event of overproduction. Because systemic toxicity is a fundamental challenge for the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies are currently being evaluated with the aim of reducing the toxic effects of this class of biological effectors while preserving their therapeutic efficacy.

[0050] Interleukin-2 (IL-2) exerts both stimulatory and regulatory functions in the immune system and, together with other members of the common gamma (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its effects by binding to the IL-2 receptor (IL-2R), which consists of either a trimeric receptor composed of the IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains, or the dimeric βγ IL-2R(1,3). Both IL-2R variants are capable of signaling upon binding to IL-2. However, the trimeric αβγ IL-2R has approximately 10- to 100-fold higher affinity for IL-2 than the dimeric βγ IL-2R(3), suggesting that CD25 confers high-affinity binding between IL-2 and 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), which are sensitive to IL-2 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 vigorously to IL-2 in vitro and in vivo.

[0051] Expression of the high-affinity IL-2R is crucial for T cell responsiveness to low concentrations of transiently available IL-2 in vivo. IL-2Rα expression is absent on naive 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 naive T cells after antigen activation. γc is much more slowly regulated and is constitutively expressed on all lymphoid cells. Once the high-affinity IL-2R is induced by antigen, IL-2R signaling upregulates IL-2Rα expression in part through Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism for maintaining high-affinity IL-2R expression and sustaining IL-2 signaling as long as a source of IL-2 remains available.

[0052] IL-2 is captured by IL-2Rα via a large hydrophobic binding surface surrounded by a polar rim, resulting in a relatively weak interaction (Kd 10-8 M) with fast binding-dissociation kinetics. However, the IL-2Rα-IL-2 binary complex induces a very small conformational change in IL-2 that facilitates its association with IL-2Rβ through distinct polar interactions between IL-2 and IL-2Rβ. As shown by Ciardelli's data, the pseudo-high affinity of the IL2 / α / β trimeric complex (i.e., Kd ∼300 pM) clearly indicates the greater stability of the trimeric complex compared to either IL2 bound to the α chain alone (Kd = 10 nM) or IL2 bound to the β chain alone (Kd = 450 nM). In either case, the IL2 / α / β trimer then recruits the γ chain into a signaling-capable quaternary complex, facilitated by the large, complex binding site on the IL2-bound β chain for the γ chain.

[0053] In other words, the ternary IL-2Rα-IL-2Rβ-IL-2 complex then recruits γc through a weak interaction with IL-2 and a stronger interaction with IL-2Rβ, generating a stable quaternary high-affinity IL-2R (Kd 10-11 M, i.e., 10 pM). Formation of the high-affinity quaternary IL-2-IL-2R complex leads to signaling by the 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, resulting in rapid degradation of IL-2, IL-2Rβ, and γc, while IL-2Rα is recycled to the cell surface (Hemar et al., 1995; Yu and Malek, 2001). Thus, functional actions requiring sustained IL-2R signaling require a continuous source of IL-2 to engage IL-2Rα to form additional IL-2-IL-2R signaling complexes.

[0054] Interleukin-15 (IL-15), another member of the four-alpha-helical bundle family of cytokines, has also emerged as an immunomodulator for the treatment of cancer. IL-15 is initially captured by IL-15Rα, which is expressed on antigen-presenting dendritic cells, monocytes, and macrophages. IL-15 exerts a wide range of actions, inducing the differentiation and proliferation of T, B, and natural killer (NK) cells by signaling through the IL-15 / IL-2-R-β (CD122) and common γ chain (CD132). It further regulates the differentiation and proliferation of CD8 + Enhances T cell cytolysis and prolongs antigen-sensitized CD8 +IL-15 induces CD44 memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces dendritic cell maturation. It does not stimulate immunosuppressive T regulatory cells (Tregs). Therefore, selective enhancement of IL-15 action in the tumor microenvironment may enhance innate and specific immunity to combat tumors (Waldmann et al., 2012). IL-15 was initially identified for its ability to stimulate T cell proliferation through common receptor components (IL-2R / 15Rβ-γc) in a manner similar to IL-2, as well as through signaling via 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, and 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 for the maintenance of forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and their retention 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. IL-2 also plays a critical role in initiating activation-induced cell death (AICD), a process that leads to the elimination of autoreactive T cells, whereas IL-15 is an anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000). IL-15 co-delivered with an HIV peptide vaccine has been shown to overcome CD4+ T cell deficiency by promoting the persistence of antigen-specific CD8+ T cells and preventing 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 - / - and IL-15 - / - This is further emphasized by the phenotype of the knockout mice, which exhibit reduced total CD8+ T cell numbers and are deficient in memory CD8+ T cells, NK cells, NK / T cells, and several subsets of intestinal intraepithelial lymphocytes, indicating that IL-5 provides essential positive homeostatic functions to these cell subsets (Lodolce et al., 1996; Kennedy et al., 1998). The similarities in the phenotypes of these two strains of knockout mice suggest the importance of IL-15Rα in maintaining physiologically relevant IL-15 signals.

[0056] IL-15 is trans-presented by the IL-15 receptor alpha chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells (Han et al., 2011). The IL-15Rα chain acts as a chaperone protein, stabilizing and enhancing IL-15 activity (Desbois et al., 2016). Exogenous IL-15 may have limited impact on cancer patients due to its dependency on IL-15Rα, which is often downregulated in cancer patients. Therefore, a fusion protein, RLI, consisting of the sushi domain of IL15Ra linked to IL-15 via a linker, was proposed as an alternative approach to IL15 therapy (Bessard et al., 2009). It was found that administration of soluble IL-15 / IL-15Rα complexes significantly enhanced the serum half-life and bioavailability of IL-15 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, regulates phagocytosis, and stimulates the secretion of IL-8 and IL-1R antagonists. It functions through activation of JAK2, p38 and ERK1 / 2 MAPKs, Syk kinase, and NF-kB transcription factors (Pelletier et al., 2002). In mast cells, IL-15 can act as a growth factor and 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 the induction of antibody responses partially independent of CD4 help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages, and dendritic cells efficiently 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 exhibit maturation with increased expression of CD83, CD86, CD40, and MHC class II, and are resistant to apoptosis and exhibit 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 can support muscle cell differentiation (Quinn et al., 1995). It stimulates muscle cells and muscle fibers to accumulate contractile proteins and can slow muscle wasting in rats with cancer-associated cachexia (Figueras et al., 2004). IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce the growth and survival of microglia (Hanisch et al., 1997).

[0059] Interleukin-7 (IL-7), also a member of the IL-2 / IL-15 family, is a well-characterized pleiotropic 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, it was described as a growth and differentiation factor for precursor B lymphocytes, but later studies demonstrated the critical involvement of IL-7 in the development and differentiation of T lymphocytes. Interleukin-7 signaling is essential for optimal CD8+ T cell function, homeostasis, and the establishment of memory (Schluns et al., 2005). al., 2000), it is required for most T cell subsets and its expression has been proposed to be important for the regulation of T cell numbers.

[0060] IL-7 is expressed by IL-7Rα and γ cIL-7Rα binds to a dimeric receptor containing IL-7Rα, forming a ternary complex that plays a fundamental role in extracellular matrix remodeling, T and B cell development, and homeostasis (Mazzucchelli and Durum, 2007). IL-7Rα also cross-reacts with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), forming a ternary complex that activates the TSLP pathway, leading to T and dendritic cell proliferation in humans and B cell development in mice (Leonard, 2002). Therefore, tight regulation of the signaling cascade activated by the complex is crucial for normal cellular function. Attenuated stimulation of the IL-7 pathway caused by mutations in the IL-7Rα ectodomain inhibits T and B cell development and results 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 reconstitution in cancer patients after cytotoxic chemotherapy. IL-7 therapy can enhance immune reconstitution and even limited thymic function by facilitating peripheral proliferation of even a small number of recent thymic emigrant cells. Therefore, IL-7 therapy may be able to restore 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) that are covalently linked to generate the so-called biologically active 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 with a linker connecting the p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays a crucial role in the early inflammatory response to infection and in generating Th1 cells, which favor cell-mediated immunity. It has been discovered that overproduction of IL-12 can be dangerous to the host as it is involved in the pathogenesis of several autoimmune inflammatory diseases (eg, MS, arthritis, type 1 diabetes).

[0063] The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of the IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T cells and natural killer cells (Trinchieri et al., 2003). IL-12Rβ1 binds to the IL-12p40 subunit, while IL-12p35 associated with IL-12Rβ2 confers intracellular signaling capabilities (Benson et al., 2011). IL-12R signaling induces the 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 effects of IL-12 are primarily mediated by 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 effects of IL-12, including the differentiation of CD4+ T cells toward a Th1 phenotype (Montepaone et al., 2014).

[0064] Regulatory T cells actively suppress immune system activation, preventing pathological self-reactivity and resulting autoimmune disease. Developing drugs and methods that selectively activate regulatory T cells for the treatment of autoimmune disease has been the subject of intense research, largely unsuccessful until the development of the present invention, which can selectively deliver active interleukins to sites 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 key role in maintaining resistance to self-antigens and regulating immune responses to foreign antigens. Several autoimmune and inflammatory diseases, including type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and graft-versus-host disease (GVHD), have been shown to have deficiencies in Treg cell numbers 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 investigated for autoimmune diseases is the transplantation of autologous ex vivo expanded Treg cells (Tang, Q., et al., 2003, Cold Spring Harb. Perspect. Med., 3:1-15). This approach has shown promise in treating animal models of disease and in some early-phase human trials, but it requires personalized treatment with the patient's own T cells, is invasive, and technically complex. Another approach is treatment with low-dose interleukin-2 (IL-2). Treg cells characteristically express high constitutive levels of the high-affinity IL-2 receptor IL2Rαβγ, which is composed of the subunits IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), and Treg cell development has been shown to be dependent on IL-2 (Malek, TR, et al., 2010, Immunity, 33:153-65).

[0066] Conversely, immune stimulation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) has been approved for treating 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] Chronic GVHD patients (Koreth, J., et al., 2011, N Engl J Clinical trials of low-dose IL-2 treatment in patients with idiopathic leukemia (N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated elevated Treg levels and signs of clinical efficacy. New clinical trials have been initiated investigating the efficacy of IL-2 in several other autoimmune and inflammatory diseases. The rationale for using so-called low-dose IL-2 was to recruit the high IL-2 affinity of the trimeric IL-2 receptor constitutively expressed on Tregs, while leaving other T cells that do not express the high-affinity receptor in an inactive state. Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, CA) is the recombinant form of IL-2 used in these trials, but it is associated with high toxicity. At high doses, aldesleukin is approved for the treatment of metastatic melanoma and metastatic kidney cancer, but its side effects are so severe that its use is recommended only 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 than many other immune cell types due to their expression of IL2Ralpha (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower doses raised safety and tolerability concerns, and the treatments used employed daily subcutaneous injections either long-term or intermittent 5-day treatment courses. Therefore, there is a need for autoimmune disease therapies that enhance Treg cell numbers and function, target Treg cells more specifically than IL-2, and are safer, more tolerable, and administered less frequently.

[0069] One proposed approach to improving the therapeutic index of IL-2-based therapy for autoimmune diseases is to use variants of IL-2 that are selective for Treg cells over other immune cells. IL-2 receptors are expressed on a variety of immune cell types, including T cells, NK cells, eosinophils, and monocytes. This widespread expression pattern may explain their pleiotropic effects on the immune system and their high systemic toxicity. In particular, activated T effector cells, like lung epithelial cells, express IL2Rαβγ. However, activating T effector cells directly counteracts the goal of attenuating and regulating the immune response, and activating lung epithelial cells leads to known dose-limiting side effects of IL-2, such as pulmonary edema. Indeed, a major side effect of high-dose IL-2 immunotherapy is vascular leak syndrome (VLS), which leads to intravascular fluid accumulation in organs such as the lungs and liver, followed by pulmonary edema and hepatocellular injury. There is no treatment for VLS other than discontinuing IL-2. Low-dose IL-2 treatment regimens have been tested in patients to avoid VLS, but at the cost of suboptimal treatment outcomes.

[0070] According to the literature, VLS is thought to be caused by the release of proinflammatory cytokines from IL-2-activated NK cells. However, there is some evidence that pulmonary edema results from direct binding of IL-2 to pulmonary endothelial cells expressing low to moderate levels of functional αβγIL-2R. Blocking IL-2 binding to CD25 using an anti-CD25 monoclonal antibody (mAb) or a CD122-specific IL-2 / anti-IL-2 mAb (IL-2 / mAb) complex in CD25-deficient host mice eliminated pulmonary edema associated with the interaction of IL-2 with pulmonary endothelial cells, thus preventing VLS.

[0071] Treatment with interleukin cytokines other than IL-2 has been more limited. IL-15 exerts immune cell stimulatory effects similar to IL-2 but lacks the same inhibitory effects, making it a promising immunotherapeutic candidate. Clinical trials of recombinant human IL-15 for the treatment of metastatic malignant 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 undergoing clinical trials to treat various forms of cancer. However, IL-15 therapy is known to be associated with undesirable adverse effects, such as aggressiveness of certain 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 naive and memory T cell homeostasis in the periphery. Furthermore, it is important for lymph node (LN) organogenesis 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 the number of regulatory T cells, as tracked by FoxP3 expression (Sportes et al., 2008). In clinical trials reported in 2006, 2008, and 2010, patients with various cancers, including metastatic melanoma or sarcoma, received subcutaneous injections of different doses of IL-7. Little toxicity was observed beyond transient fever and mild erythema. Circulating levels of CD4+ and CD8+ T cells significantly increased, while the number of Tregs decreased. The diversity of the TCR repertoire increased after IL-7 therapy. However, the antitumor activity of IL-7 has not been fully evaluated (Gao et al., 2015). Results suggest that IL-7 therapy may enhance and expand immune responses.

[0073] IL-12 is a pleiotropic cytokine whose actions create an interconnection between innate and adaptive immunity. IL-12 was first described as a factor secreted by PMA-induced EBV-transformed B cell lines. Based on its actions, IL-12 has been named a cytotoxic lymphocyte maturation factor and natural killer cell-stimulating factor. IL-12 appeared to be an ideal candidate for human tumor immunotherapy because it bridges the gap between innate and adaptive immunity and potently stimulates the production of IFNγ, a cytokine that orchestrates natural anti-cancer defense mechanisms. However, severe side effects associated with systemic administration of IL-12 in clinical trials and the cytokine's very narrow therapeutic index have significantly dampened enthusiasm for its use in cancer patients (Lasek et al., 2014). Targeting IL-12 delivery to tumors, which may alleviate some of the traditional problems associated with IL-12 therapy, is currently being investigated for cancer.

[0074] The direct use of IL-2 as an agonist that binds to IL-2R and therapeutically modulates immune responses is problematic due to well-documented therapeutic risks, such as its short serum half-life and high toxicity. These risks have also limited the therapeutic development and use of other cytokines. New forms of cytokines that mitigate these risks are needed. Disclosed herein are compositions and methods, including IL-2 and IL-15, as well as other cytokines, functional fragments and muteins of cytokines, variants and subunits of cytokines, and conditionally active cytokines, designed to address these risks and provide needed immunomodulatory therapeutics.

[0075] The present invention is designed to address the shortcomings of direct IL-2 therapy and other cytokine-based therapies, for example, using cytokine blocking moieties, e.g., steric blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, linking polypeptides, e.g., 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 (IFNs, including IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxin), transforming growth factors (e.g., TGF-beta1, TGF-beta2, TGF-beta3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), are highly potent when administered to patients. As used herein, "chemokines" refers to a family of small cytokines capable of inducing directional chemotaxis in nearby responder cells. While cytokines can provide powerful therapeutics, they are associated with undesirable effects that are difficult to control clinically and have limited their clinical use. The present disclosure relates to novel forms of cytokines that can be used in patients with reduced or eliminated undesirable effects. In particular, the present disclosure relates to pharmaceutical compositions comprising chimeric polypeptides (fusion proteins), nucleic acids encoding the fusion proteins, and pharmaceutical formulations thereof containing cytokines or active fragments or muteins of cytokines that have reduced cytokine receptor activating activity relative to the corresponding cytokine. Nevertheless, under selected conditions or in a selected biological environment, the chimeric polypeptides often activate their cognate receptors with the same or greater potency than the corresponding naturally occurring cytokine. As described herein, this is typically accomplished using a cytokine-blocking moiety that blocks or inhibits the receptor activating function of the cytokine, its active fragment, or mutein under normal conditions, but not under selected conditions, such as those present at the site of desired cytokine action (e.g., a site of inflammation or a tumor).

[0076] Chimeric polypeptides and nucleic acids encoding them can be made using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be made using recombinant DNA technology, synthetic chemistry, or a combination of these techniques, and expressed in a suitable expression system, such as CHO cells. Chimeric polypeptides can also be made by expressing suitable nucleic acids using, for example, synthetic or semi-synthetic chemical technology. In some embodiments, a blocking moiety can be attached to a cytokine polypeptide by sortase-mediated conjugation. A "sortase" is a peptidyltransferase that modifies proteins by recognizing and cleaving carboxyl-terminal sorting signals embedded in or attached to the terminus of a target protein or peptide. Sortase A catalyzes the cleavage of an 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 transient binding of the Thr residue to an active site Cys residue on the enzyme to form an enzyme-thioacyl intermediate. To complete the transpeptidation and create the peptide-monomer conjugate, a biomolecule bearing an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displacing sortase A and joining the two molecules together.

[0077] To form a cytokine blocking moiety fusion protein, a cytokine polypeptide is first tagged at the N-terminus with a polyglycine sequence or at the C-terminus with an LPXTG motif (SEQ ID NO: 442). The blocking moiety or other element has attached to it a respective peptide that serves as an acceptor site for the tagged polypeptide. For conjugation via its N-terminus to a domain bearing an attached LPXTG motif (SEQ ID NO: 442) acceptor peptide, the polypeptide would be tagged with an N-terminal stretch of polyglycines. For conjugation via its C-terminus to a domain bearing a polyglycine peptide, the polypeptide would be tagged at its C-terminus with the LPXTG (SEQ ID NO: 442) sortase recognition sequence. Sortase recognizes the polyglycine and LPXTG (SEQ ID NO: 442) sequences and forms a peptide bond between the polymer-peptide and the tagged polypeptide. The sortase reaction discards glycine residues as intermediates and occurs at room temperature.

[0078] Various mechanisms can be employed to eliminate or reduce the inhibition provided by a blocking moiety. For example, a pharmaceutical composition can include a cytokine moiety and a blocking moiety, e.g., a steric blocking moiety, with a protease-cleavable linker containing a protease cleavage site located between the cytokine and the cytokine blocking moiety or within the cytokine blocking moiety. Upon cleavage of the protease cleavage site, the blocking moiety can dissociate from the cytokine, after which the cytokine can activate the cytokine receptor. The cytokine moiety can also be blocked by a specific blocking moiety, such as an antibody, that binds to an epitope found on the cytokine of interest.

[0079] Any suitable linker can be used, for example, the linker can be glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 443) or (Gly3Ser) n, (SEQ ID NO: 444), where n is 1, 2, 3, 4, or 5. Typically, the sortase recognition motif comprises 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 attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of the inhibitor or other domain. In other embodiments, there is a covalent linkage between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of the inhibitor or other domain.

[0080] Thus, as described in detail herein, the cytokine blocking moiety used may be a steric blocker. As used herein, a "steric blocker" refers to a polypeptide or polypeptide moiety that may be covalently attached 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), but is otherwise not covalently attached to the cytokine polypeptide. A steric blocker may be non-covalently attached 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. Steric inhibition of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocker, e.g., by enzymatic cleavage of a fusion protein containing the steric blocker and cytokine peptide at a site between the steric blocker and the cytokine polypeptide.

[0081] As described in more detail herein, the blocking function may be combined with or due to the presence in the pharmaceutical composition of additional functional components such as targeting domains, serum half-life extending elements, and protease-cleavable linking polypeptides. For example, the serum half-life extending polypeptide may also be a steric blocking agent.

[0082] To provide a concise disclosure of the overall scope of the invention, the cytokine IL-2 will be used as an exemplary cytokine to describe aspects of the invention in detail. However, the invention and disclosure are not limited to IL-2. It will be apparent to those skilled in the art that the disclosure, including the methods, polypeptides, and nucleic acids of the disclosure, fully describes and enables the use of other cytokines, fragments, variants, cytokine subunits, and muteins, 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 functional fragments or muteins of any of the foregoing. Preferred cytokines for use in the fusion proteins disclosed herein are IL-2, IL-12, IFN alpha, IFN beta, IFN gamma, and muteins, functional variants, and functional fragments or subunits of any of the foregoing. For example, the cytokine IL-12 cytokine can be a p35 subunit, p40 subunit, heterodimer.

[0083] Various factors ensure delivery and activity of IL-2 at the desired site of IL-2 activity, and systemic exposure to the interleukin is severely limited by a blocking and / or targeting strategy preferentially associated with a serum half-life extension strategy in which the blocked form of the interleukin circulates for an extended period of time (preferably 1-2 weeks or more), while the activated form has the typical serum half-life of an interleukin.

[0084] In comparison with the extended serum half-life forms, the serum half-life of intravenously administered IL-2 is only about 10 minutes due to distribution into the large systemic extracellular space, which is approximately 15 L in an average-sized adult. IL-2 is then metabolized by the kidney with a half-life of approximately 2.5 hours (Smith, K. "Interleukin 2 immunotherapy." Therapeutic Immunology 240 (2001)). Other measurements have shown that IL-2 has a very short plasma half-life of 85 minutes after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, G.I., et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of the invention, the half-life extending element is linked to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammation- or tumor-specific protease) to release the full action of the interleukin at the desired site and separate it from the extended half-life of the uncleaved form. In such embodiments, the fully active and free interleukin will have very different pharmacokinetic (pK) properties—half-lives of hours instead of weeks. Additionally, exposure to the active cytokine is limited to the desired site of cytokine action (e.g., the site of inflammation or tumor), reducing systemic exposure to the active cytokine and associated toxicity and side effects.

[0085] Other cytokines contemplated in the present invention have pharmacology similar to IL-2 (e.g., IL-15, 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 shortcomings of using these agents directly, providing chimeric polypeptides that may have an extended half-life and / or may be directed to a desired site of activity (e.g., sites of inflammation or tumors).

[0086] Optionally, IL-2 can be engineered to bind to the IL-2R complex as a whole or specifically to one of the three IL-2R subunits with an affinity that differs from that corresponding to wild-type IL-2, e.g., to selectively activate Tregs or Teffs. For example, an IL-2 polypeptide that is said to have a higher affinity for the trimeric form of the IL-2 receptor than for the dimeric beta / gamma form of the IL-2 receptor relative to wild-type IL-2 can have an amino acid sequence that includes one of the following sets of mutations relative to SEQ ID NO: 1 (the mature IL-2 protein comprising amino acids 21-153 of human IL-2 having Uniprot Accession No. P60568-1): (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K64R, V69A, and Q74P; 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 technique can also be applied to prepare muteins 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 factors (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, muteins can be prepared that have the desired binding affinity for their cognate receptors.

[0087] As noted above, any of the mutant IL-2 polypeptides disclosed herein can include the sequences described; they can also be limited to the sequences described and other sequences identical to SEQ ID NO: 1. Furthermore, any of the mutant IL-2 polypeptides disclosed herein can optionally include a substitution of the cysteine ​​residue at position 125 of SEQ ID NO: 1 with another residue (e.g., serine) and / or a 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 pharmacokinetics of the molecule to maximize Treg cell activation. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro required exposure to effective concentrations of IL-2 for a minimum of 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 after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). Due to its short half-life, maintaining circulating IL-2 at levels above those required to stimulate T cell proliferation for the required duration requires high doses or frequent administration, resulting 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 a domain that has a longer circulating half-life than IL-2 and allows binding to the FcRn receptor, may achieve target drug concentrations over a specific period at lower doses and lower peak levels than IL-2. Therefore, such IL-2 analogs may require lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of IL-2 drugs may also be more tolerable to patients. Therapeutic agents with these characteristics may clinically lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance with therapy. Alternatively, IL-2 or a mutein of IL-2 (referred to herein as "IL-2 * ") can be selectively directed to the intended site of action (e.g., a site of inflammation or a tumor). This targeting can be achieved by one of several strategies, including the addition of a domain to the administered agent that contains a blocking agent of truncated IL-2 (or a mutein), or by a targeting domain, or by a combination of both.

[0089] In some embodiments, IL-2 is adapted to have higher or lower affinity depending on the desired target. * Partial agonists can be made, e.g., IL-2 *can be engineered to bind with enhanced affinity to one receptor subunit but not to the others. These classes of partial agonists, unlike full agonists or full antagonists, offer the ability to tailor signaling properties to elicit desired functional properties while not meeting a threshold of undesirable properties. Given the differential activity of partial agonists, the repertoire of IL-2 variants may be engineered to exert an even finer degree of unique signaling activity, ranging from near-full-to-partial agonism to full antagonism.

[0090] In some embodiments, IL-2 * has an altered affinity for IL-2Rα. In some embodiments, IL-2 * has a higher affinity for IL-2Rα than wild-type IL-2. * In one embodiment, the IL-2 * In another embodiment, an IL-2Rα-binding domain is selected from IL-2Rβ- and IL-2Rγ-binding domains that exhibit enhanced binding affinity to IL-2Rβ, e.g., the N-terminus of IL-2Rβ, thereby eliminating the functional requirement for IL-2Rα. In another embodiment, an IL-2Rα-binding domain is selected from IL-2Rβ- and IL-2Rγ-binding domains that exhibit increased binding affinity to IL-2Rβ but reduced binding to IL-2Rγ, thereby impairing IL-2Rβγ heterodimerization and signaling. * is generated.

[0091] Blocking moieties, as described in more detail below, can also be used to favor binding to or activation of one or more receptors. In one embodiment, a blocking moiety is added such that IL-2Rβγ binding or activation is blocked but IL-2Rα binding or activation is unchanged. In another embodiment, a blocking moiety is added such that IL-2Rα binding or activation is attenuated. In another embodiment, a blocking moiety is added such that binding to or activation of all three receptors is inhibited. This block can be relieved by removal of the blocking moiety in certain circumstances, for example, by proteolytic cleavage of a linker connecting one or more blocking moieties to the cytokine.

[0092] Similar approaches can be applied to improving other cytokines, particularly for use as immune stimulators, e.g., 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) can be tailored to maximize activation of effector cells (e.g., effector T cells, NK cells) and / or cytotoxic immune response-promoting cells (e.g., inducing dendritic cell maturation), preferably not systemically, but at the desired site of action, e.g., tumor.

[0093] Thus, provided herein are pharmaceutical compositions comprising at least one cytokine polypeptide, such as an interleukin (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), an interferon (IFN, including IFN-alpha, IFN-beta, and IFN-gamma), a tumor necrosis factor (e.g., TNF-alpha, lymphotoxin), a transforming growth factor (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3), a chemokine (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage-colony-stimulating factor (GM-CS), or a functional fragment or mutein of any of the above. The polypeptide typically also comprises at least one linker amino acid sequence, which, in certain embodiments, is capable of being cleaved by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (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 contains a blocking moiety, e.g., a steric blocking polypeptide moiety, capable of blocking the action of the interleukin polypeptide. The blocking moiety can comprise, for example, a human serum albumin (HSA) binding domain or polyethylene glycol (PEG), optionally branched or multiarmed. Alternatively, the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof and a blocking moiety, e.g., a steric blocking polypeptide moiety, which blocks the action of the cytokine polypeptide on a cytokine receptor, and which, in some embodiments, comprises a protease-cleavable domain. In some embodiments, blocking or reducing cytokine action is achieved simply by attaching an additional domain with a very short linker to the N- or C-terminus of the interleukin domain. In such embodiments, it is expected that protease digestion of the blocking moiety or of the short linker tethering the blocking agent to the interleukin will release the block.Once the domain is sheared or released, it can no longer effect blocking of cytokine action.

[0094] Pharmaceutical compositions, e.g., chimeric polypeptides, can contain two or more cytokines, which can be the same or different cytokine polypeptides. For example, the two or more different cytokines 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 cytokine polypeptides has the function of regulating the function of the other cytokine polypeptide. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine polypeptide is T cell stimulatory and the second cytokine polypeptide is non-T cell stimulatory. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine is a chemoattractant, e.g., CXCL10, and the second cytokine is an immune cell stimulator.

[0095] Preferably, the cytokine polypeptides (including functional fragments) contained in the fusion proteins disclosed herein have not been mutated or engineered to alter the properties of the naturally occurring cytokine, including receptor binding affinity and specificity or serum half-life. However, amino acid sequence changes from the naturally occurring cytokine (including wild-type) are acceptable, for example, to facilitate cloning and achieve desired expression levels.

[0096] blocking part A blocking moiety can be any moiety that inhibits the cytokine's ability to bind to and / or stimulate its receptor. A blocking moiety can inhibit the cytokine's ability to bind to and / or stimulate its receptor by sterically blocking the cytokine and / or by non-covalently binding to it. Examples of suitable blocking moieties include full-length or cytokine-binding fragments or muteins of the cytokine's cognate receptor. Antibodies and fragments thereof, including 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 variable domains of camelid nanobodies (VHH), dAbs, etc., that bind to cytokines can also be used. Other suitable antigen-binding domains that bind to cytokines can also be used, including non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of cytokines to their cognate receptors. Advantageously, such moieties can also function as half-life extension elements. For example, peptides modified by conjugation with water-soluble polymers such as PEG can sterically inhibit or prevent 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, etc., as well as fragments and muteins of such polypeptides, may also be used. For example, antibodies and antigen-binding domains that bind to proteins with long serum half-lives, such as HSA, immunoglobulins or transferrin, or that bind to receptors that recycle to the plasma membrane, such as FcRn or transferrin receptor, can also inhibit cytokines, particularly when bound to their 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 variable domains of camelid nanobodies (VHH), dAbs, etc. Other suitable antigen-binding domains that bind cytokines may also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds.

[0097] In an illustrative example, if IL-2 is the cytokine in the chimeric polypeptide, the blocking moiety can be the full-length or fragment or mutein of the IL-2 receptor alpha chain (IL-2Rα) or IL-2 receptor beta (IL-2Rβ) or gamma chain (IL-2Rγ), an anti-IL-2 single-domain antibody (dAb) or scFv, Fab, an anti-CD25 antibody or fragment thereof, and an anti-HSA dAb or scFv, etc. As further described herein, if an antibody fragment is used to attenuate the action of the cytokine polypeptide, the blocking moiety in the fusion protein can be a single-chain antibody-binding fragment, e.g., an scFv. The blocking moiety can also be one half of a two-chain antigen-binding fragment, e.g., a VH-CH1 that associates with a complementary VL-CL on a second polypeptide to form an antibody binding site that binds to the cytokine polypeptide.

[0098] In vivo half-life extension element Preferably, the chimeric polypeptide comprises an in vivo half-life extending element. Increasing the in vivo half-life of a therapeutic molecule with a naturally short half-life allows for more tolerable and manageable dosing regimens without sacrificing efficacy. As used herein, a "half-life extending element" is a portion of a chimeric polypeptide that increases its in vivo half-life and improves its pK, for example, by altering its size (to exceed the renal filtration cutoff), shape, hydrodynamic diameter, charge, or parameters of absorption, biodistribution, metabolism, and excretion. An exemplary method for improving the pK of a polypeptide is by expressing an element in the polypeptide chain that binds to receptors that recycle to the plasma membrane of cells rather than being degraded in lysosomes, such as the FcRn receptor and transferrin receptor on endothelial cells. Three proteins, such as human IgG, HSA (or fragments), and transferrin, persist in human serum for much longer than would be predicted by their size alone, which is a function of their ability to bind to receptors that recycle rather than be degraded in lysosomes. These proteins or their fragments that retain FcRn binding ability are routinely linked to other polypeptides to extend their serum half-life. In one embodiment, the half-life extending element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may be directly bound to the pharmaceutical composition or may be bound via a short linker. Fragments of HSA may also be used. HSA and its fragments can function as both blocking moieties and half-life extending elements. 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, transferrin, etc. Examples of such polypeptides include polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), dAbs, and fragments thereof. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0100] Some preferred serum half-life extending elements are polypeptides comprising complementarity-determining regions (CDRs) and optional non-CDR loops. Beneficially, such serum half-life extending elements can extend the serum half-life of cytokines and can also function as cytokine inhibitors (e.g., by steric blocking, non-covalent interactions, or a combination thereof) and / or as targeting domains. In some cases, the serum half-life extending element is a domain derived from an immunoglobulin molecule (Ig molecule) or from an engineered protein scaffold that mimics antibody structure and / or binding activity. Ig can be of any class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of an Ig molecule folds into a series of parallel beta strands connected by loops. In the variable region, three of the loops constitute "complementarity-determining regions" (CDRs), which determine 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, inter-connected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions are further subdivided into regions of hypervariability, termed complementarity-determining regions (CDRs), which are highly variable in sequence and / or involved in antigen recognition and / or form mostly structured loops, interspersed with more conserved regions termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of the binding moieties described herein. The variable domains of immunoglobulin molecules have several beta strands arranged in two sheets.The variable domains of both immunoglobulin heavy and light chains contain three hypervariable loops or complementarity-determining regions (CDRs). The three CDRs of a V domain (CDR1, CDR2, CDR3) are clustered at one end of a beta barrel. The CDRs are the loops connecting beta strands BC, C'-C", and FG of the immunoglobulin fold, while the lower loops connecting beta strands AB, CC', C"-D, and EF of the immunoglobulin fold, and the upper loop connecting the DE strand of the immunoglobulin fold, are non-CDR loops. In some embodiments of the present disclosure, at least some 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: the AB, CD, EF, and DE loops of the C1 class domain of an Ig or Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of the C2 class domain of an Ig or Ig-like molecule; and the DE, BD, GF, A(A1A2)B, and EF loops of the I (intermediate) class domain of an Ig or Ig-like molecule.

[0101] In the variable domain, CDRs are considered to be responsible for antigen recognition and binding, while FR residues are considered to be a scaffold for the CDRs. However, in some cases, some FR residues play an important role in antigen recognition and binding. Framework region residues that affect Ag binding are divided into two categories. The first category is FR residues that contact the antigen and are therefore part of the binding site, and some of these residues are located close to the CDRs in sequence. Other residues are located far from the CDRs but are in close proximity to them in the three-dimensional structure of the molecule, for example, in the loops of the heavy chain.

[0102] The binding moiety can be any type of polypeptide. For example, in some cases, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered large serum protein. Large 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 scaffold (as proposed in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine-knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin binding module, or DNA or RNA aptamer scaffold.

[0103] In some cases, the serum half-life extending element comprises a binding site for a large serum protein. In some embodiments, the CDRs provide the binding site for the large serum protein. The large serum protein is, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDRs form a binding site for an immunoglobulin light chain, such as an Igκ-free light chain or an Igλ-free light chain.

[0104] The serum half-life extending element can 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 moiety is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived nanobody (VHH). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimic, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, and monobodies.

[0105] In other embodiments, the serum half-life extending element 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 include any non-peptidic, water-soluble poly(ethylene oxide). The term "PEG" also refers to a polymer containing a majority, i.e., greater than 50%, of -OCH2CH2- repeating subunits. With respect to specific configurations, PEG can have any number of different molecular weights and structures or geometries, such as "branched," "linear," "forked," and "multifunctional," as described in more detail below. PEG is not limited to a particular structure and may be linear (e.g., end-capped, e.g., alkoxy PEG, or bifunctional PEG), branched or multi-armed (e.g., forked PEG, or PEG attached to a polyol core), or dendrimeric (or star) structures, each with or without 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 tripolymers, random tripolymers, and block tripolymers. PEG can be conjugated to polypeptides and peptides by any suitable method. Typically, a reactive PEG derivative, such as N-hydroxysuccinamidyl ester PEG, is reacted with a peptide or polypeptide containing an amino acid with a side chain containing an amine, sulfhydryl, carboxylic acid, or hydroxyl functional group, such as cysteine, lysine, asparagine, glutamine, threonine, threonine, threonine, threonine, serine, aspartic acid, or glutamic acid.

[0106] Targeting and Retention Domains For certain applications, it may be desirable to maximize the amount of time a construct is present in its desired location in the body. This can be accomplished by including an additional domain in the chimeric polypeptide (fusion protein) to affect its trafficking within the body. For example, the chimeric nucleic acid can encode a domain that directs the polypeptide to a location in the body, such as a tumor cell or an inflammatory site; this domain is referred to as a "targeting domain"; and / or encodes a domain that retains the polypeptide at a location in the body, such as a tumor cell or an inflammatory site; this domain is referred to as a "retention domain." In some embodiments, a domain can function as both a targeting and retention domain. In some embodiments, the targeting and / or retention domain is specific for a protease-rich environment. In some embodiments, the encoded targeting and / or retention domain is specific for regulatory T cells (Tregs), e.g., targeting the CCR4 or CD39 receptor. Other suitable targeting and / or retention domains include those with cognate ligands that are overexpressed in inflamed tissues, such as the IL-1 receptor or the IL-6 receptor. In other embodiments, suitable targeting and / or retention domains include those with cognate ligands that are overexpressed in tumor tissue, such as Epcam, CEA, or mesothelin. In some embodiments, the targeting domain is linked to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammatory or cancer-specific protease), thereby releasing the full action of the interleukin at the desired site. In some embodiments, the targeting 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 inflammatory or cancer-specific protease), thereby allowing the cytokine to remain at the 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 useful for targeting and retention to 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 containing two targeting 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 targeting and / or retention domains include antigen-binding domains, e.g., antibodies and fragments thereof, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies, e.g., heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH), dAbs, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic antibody binding and / or structure, e.g., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, finomers, kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, e.g., SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding portions of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0109] In some embodiments, the targeting and / or retention domain specifically binds to a cell surface molecule. In some embodiments, the targeting and / or retention domain specifically binds to a tumor antigen. In some embodiments, the targeting polypeptide specifically and independently binds to a tumor antigen selected from at least one of fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 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 targeting 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] The targeting and / or retention antigen may be a tumor antigen 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 These 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] The targeting and / or retention antigen can be an immune checkpoint protein, including, but 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] The targeting and / or retention antigen may be a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the targeting and / or retention antigen is present on tumor cells, virus-infected cells, bacteria-infected cells, damaged red blood cells, arterial plaque cells, or inflamed or fibrotic tissue cells. The targeting and / or retention antigen may comprise an immune response modifier. 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] The targeting and / or retention antigen may be a cytokine receptor. Examples of cytokine receptors include type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, and TPO receptor; type II cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), and type II IL receptor; chemokine receptors, such as CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, and XC chemokine receptor. tumor necrosis receptor superfamily receptors, such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b; TGF-beta receptors, such as TGF-beta receptor 1 and TGF-beta receptor 2; Ig superfamily receptors, such as IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), and SCFR, but are not limited to these.

[0114] Linker As described above, the pharmaceutical composition includes one or more linker sequences. The linker sequence serves to provide flexibility between polypeptides, for example, so that the blocking moiety can block the action of the cytokine polypeptide. The linker sequence can be located between any or all of the cytokine polypeptide, serum half-life extending element, and / or blocking moiety. As described herein, at least one of the linkers is protease-cleavable and contains cleavage site(s) for the desired protease(s). Preferably, the desired protease(s) is / are abundantly present or selectively expressed at the desired cytokine site of action (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the desired cytokine site of action.

[0115] Suitable linkers may vary in length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 1 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and may 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 will be recognized that the orientation of the components of a pharmaceutical composition is generally a matter of structural choice, and that multiple orientations are possible and all are intended to be encompassed by the present disclosure. For example, the blocking moiety may be located at the C-terminus or N-terminus of the cytokine polypeptide.

[0117] Proteases known to be associated with diseased cells or tissues include serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin-like proteases, actinidain, bromelain, calpain, caspases, caspase-3, and Mir. These include, but are not limited to, l-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidases, metalloendopeptidases, matrix metalloproteinases (MMPs), 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-α), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). The protease capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequence provided herein can be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), a disintegrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP can be, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9).

[0118] Proteases useful in the methods disclosed herein are shown in Table 1, with exemplary proteases and their cleavage sites shown in Table 1a: [Table 1-1] [Table 1-2] [Table 1a-1] [Table 1a-2] [Table 1a-3] [Table 1a-4]

[0119] Pharmaceutical compositions comprising the polypeptide sequences are provided herein. It is understood that, as with all peptides, polypeptides, and proteins, including fragments, additional modifications may occur in the amino acid sequence of the chimeric polypeptide (amino acid sequence variants) that do not alter 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 a desired function. The compositions comprise 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, or CCL21, a blocking moiety, e.g., a steric blocking polypeptide, and an optional serum half-life extending element, and an optional targeting polypeptide, with one or more linkers connecting each polypeptide in the composition. A first polypeptide, e.g., an IL-2 mutein, is provided to serve as an active agent. The blocking moiety is provided to block the action of an interleukin. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed in the intended target of the active agent. Optionally, the blocking moiety blocks the action of the first polypeptide by binding to the interleukin polypeptide. In some embodiments, the blocking moiety, e.g., a steric blocking peptide, is linked to the interleukin via a protease-cleavable linker that is cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full effect of the cytokine at the desired site.

[0121] The protease cleavage site may be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site. The artificially engineered protease cleavage site may be cleaved by more than one protease specific to the desired environment in which cleavage will occur, such as a tumor. The protease cleavage site may be cleavable 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 a glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 443) or (Gly3Ser) n(SEQ ID NO:444), where n is 1, 2, 3, 4, or 5. In one embodiment, the sortase recognition motif comprises 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 attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of the blocking moiety or other moiety. In one embodiment, the covalent linkage is between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of the blocking moiety or other moiety.

[0123] Cleavability and inducibility As described herein, the action of the cytokine polypeptide in the context of the fusion protein is attenuated, and protease cleavage at the desired site of action, e.g., the tumor microenvironment, liberates a form of cytokine from the fusion protein that is significantly more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine receptor agonist activity of the fusion polypeptide can be at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold less than the cytokine receptor agonist activity of the 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 the cytokine polypeptide, is substantially free of additional amino acids, and is not associated (covalently or noncovalently) with other molecules. Optionally, the cytokine polypeptide as a separate molecular entity can include some additional amino acid sequence, such as a tag or short sequence useful for expression and / or purification.

[0124] In other examples, the cytokine receptor activating (agonistic) effect of the fusion polypeptide is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold greater than the cytokine receptor activating effect of a polypeptide containing a cytokine polypeptide generated by cleavage of a protease-cleavable linker in the fusion protein. In other words, the cytokine receptor activating (agonistic) effect of a polypeptide containing a cytokine polypeptide generated by cleavage of a protease-cleavable linker in the fusion protein is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold greater than the cytokine receptor activating effect of the fusion protein.

[0125] Polypeptide Variants and Amino Acid Substitutions The polypeptides described herein can contain components (e.g., cytokines, blocking moieties) that can have the same amino acid sequence as the corresponding naturally occurring protein (e.g., IL-2, IL-15, HSA) or that have a different amino acid sequence from the naturally occurring protein, so long as the desired function is maintained. It is understood that one way of defining any known or potential modifications and derivatives of the proteins of the present disclosure and the nucleic acids encoding them is by defining sequence variants in terms of identity to a particular known reference sequence. Specifically, disclosed are polypeptides and nucleic acids 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, or 99 percent identity to the chimeric polypeptides provided herein. For example, provide polypeptide or nucleic acid that has 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% identity with any of the nucleic acid or polypeptide sequences described herein.Those skilled in the art can easily understand how to determine the identity of two polypeptides or two nucleic acids.For example, identity can be calculated after aligning two sequences so that identity is at its highest level.

[0126] Alternative methods for calculating identity can be performed using published algorithms. Optimal alignment of sequences for comparison can be performed using 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 search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), 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] The same type of identity for nucleic acids can be obtained by algorithms disclosed in, for example, Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference at least for their material regarding nucleic acid alignment. It is understood that any of the methods may typically be used, and that one of skill in the art will understand that, although the results of these various methods may differ in some cases, if identity is found by at least one of these methods, the sequence will have the specified identity and may be said to be disclosed herein.

[0128] Protein modifications include amino acid sequence modifications. Modifications in amino acid sequences can occur naturally as allelic variations (e.g., due to genetic polymorphisms), can occur due to environmental influences (e.g., exposure to ultraviolet light), or can be generated by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point, deletion, insertion, and substitution mutations. These modifications can result in changes in the amino acid sequence, silent mutations, altered 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-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions will usually be smaller than amino- or carboxyl-terminal fusions, e.g., insertions of about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, deletions of about 2 to 6 residues or less are made at any one site in the protein molecule. Amino acid substitutions are typically single residue substitutions, but can occur at several different locations at once; insertions will usually be on the order of about 1-10 amino acid residues; deletions will range from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., a deletion of two residues or an insertion of two residues. Substitutions, deletions, insertions, or any combination thereof, can be combined to arrive at the final construct. Mutations should not place the sequence outside the reading frame and preferably do not create complementary regions that could result in secondary mRNA structure. Substitutional modifications involve 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] The modifications, including specifically recited amino acid substitutions, are made by known methods. For example, modifications can be made by site-specific mutagenesis of nucleotides in the DNA encoding the polypeptide to thereby generate DNA encoding the modification, followed by expression of the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis.

[0130] Modifications can be selected to optimize binding. For example, affinity maturation techniques can be used to alter the binding properties of scFvs by introducing random mutations into the complementarity-determining regions (CDRs). Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens, or error-prone PCR. Multiple rounds of mutation and selection can be performed, for example, using phage display.

[0131] The present disclosure also relates to nucleic acids encoding the chimeric polypeptides described herein, and the use of such nucleic acids to produce the chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) that encode the chimeric polypeptides, and therapeutic uses of such DNA and RNA molecules.

[0132] Exemplary Compositions Exemplary fusion proteins of the invention combine the above elements in various orientations. The orientations described in this section are intended as examples and should not be considered limiting.

[0133] In some embodiments, the fusion protein comprises a cytokine, a blocking moiety, and a half-life extending element. In some embodiments, the cytokine is disposed between the half-life extending element and the blocking moiety. In some embodiments, the cytokine is N-terminal to the blocking moiety and the half-life extending element. In some such embodiments, the cytokine is proximal to the blocking moiety; in some such embodiments, the cytokine is proximal to the half-life extending element. In all embodiments, at least one protease-cleavable linker must be included so that the cytokine can be activated upon cleavage. In some embodiments, the cytokine is C-terminal to the blocking moiety and the half-life extending element. Additional elements may be attached by a cleavable linker, a non-cleavable linker, or by direct fusion.

[0134] In some embodiments, the blocking domain used is capable of extending half-life, and the cytokine is positioned between two such blocking domains, hi some embodiments, the cytokine is positioned between two blocking domains, one of which is capable of extending half-life.

[0135] In some embodiments, two cytokines are included in the same construct. In some embodiments, the cytokines are each linked to two blocking domains (for a total of three in one molecule), with one blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extending domains may be included to optimize pharmacokinetic properties. In some cases, it is beneficial to include two of the same cytokine 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, and in some embodiments, the third cytokine may function to block the other two in place of a 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 (e.g., scFv, dAb) that binds to serum albumin, human or humanized IgG, or a fragment of any of the above. In some preferred embodiments, the blocking 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 binding and activation of the cytokine receptor, another cytokine, or a fragment of any of the above. In preferred embodiments including an additional targeting domain, the targeting domain is an antibody that binds to a cell surface protein that is abundant on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin.

[0138] In an embodiment, the fusion protein may contain an IL-2 polypeptide. The fusion protein containing an IL-2 polypeptide may comprise or consist of any one of the amino acid sequences set forth in 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 herein referred to as ACP289-ACP292, ACP296-ACP302, WW0301, ACP304-ACP306, ACP309-ACP313, WW0304, WW0305, WW0306, WW0307, ​​WW0308, WW0310, WW0311, WW0312, WW0313, WW0314, WW0315, WW0316, WW0317, WW0318, WW0319, WW0320, WW0321, WW0322, WW0323, WW0324, WW0325, WW0326, WW0327, WW0328, WW0329, WW0330, WW0331, WW0332, WW0333, WW0334, WW0335, WW0336, WW0337, WW0338, WW0339, WW0340, WW0341, WW0342, WW0343, WW0344, WW0345, WW0346, WW0348, WW0349, WW0350, WW0351, WW0352, WW0353, WW0354, WW0355, WW0 ACP427 to ACP438, and ACP448 to ACP450 are also referred to as ACP414, ACP336 to ACP398, WW0472 to WW0477, ACP406 to ACP426, ACP439 to ACP447, ACP451 to ACP471, WW0729, WW0734 to WW0792, ACP101, ACP293 to ACP295, ACP316 to ACP335, ACP427 to ACP438, and ACP448 to ACP450. For example, the fusion protein may comprise the amino acid sequence of SEQ ID NO: 272. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 286. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 362. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 336. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 348. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 363. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 580.

[0139] In embodiments, a fusion protein may contain an IL-12 polypeptide. Fusion proteins containing an IL-12 polypeptide may comprise or consist of the amino acid sequence of any one of SEQ ID NOs: 368-371, 434-440, 453-519, or 523-538. 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, a fusion protein may comprise the amino acid sequence of SEQ ID NO: 459. A fusion protein may comprise the amino acid sequence of SEQ ID NO: 466. A fusion protein may comprise the amino acid sequence of SEQ ID NO: 484. The fusion protein may comprise 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. Fusion proteins containing an IFN polypeptide may comprise or consist of the amino acid sequence of SEQ ID NOs: 421-430 and 539-578. The fusion proteins disclosed as SEQ ID NOs: 421-430 and 539-578 may also 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 comprise the amino acid sequence of SEQ ID NO: 421. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 428. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 541. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 558. The fusion protein may comprise the amino acid sequence of SEQ ID NO: 577.

[0141] In some embodiments, the fusion polypeptides disclosed herein can be covalently or non-covalently linked to a second polypeptide chain. For example, the fusion polypeptide can dimerize (i.e., form a dimer), or a portion of the fusion polypeptide can associate with another polypeptide, resulting in the formation of a functional binding site for, for example, a cytokine polypeptide or serum albumin. In certain embodiments, the second polypeptide chain and the blocking moiety on the fusion polypeptide are complementary and together form a functional binding site with specificity for the cytokine polypeptide contained in the fusion polypeptide. An exemplary functional binding site that can be formed by the blocking moiety of the fusion polypeptide and a complementary second polypeptide includes the antigen-binding site of an antibody, e.g., an Fab fragment or portion thereof. For example, one chain of the Fab that binds to the cytokine can be the blocking moiety of the fusion polypeptide, e.g., VH-CH1, and the complementary VL-CL can be part of the second polypeptide. In such cases, the blocking portion of the fusion protein, i.e., VH-CH1, and a second polypeptide comprising a complementary VL-CL can associate to form a functional binding site with specificity for a cytokine polypeptide (e.g., IL-2, IL-12, IFN alpha, IFN beta) contained in the fusion protein, thereby attenuating cytokine polypeptide activity. At least a portion of the blocking portion can be on the second polypeptide chain and can include at least a portion of the blocking portion associated with the blocking portion on the fusion polypeptide.

[0142] In embodiments, a fusion protein containing an IL-2 cytokine polypeptide may be covalently or noncovalently linked to a 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 NO: 263, 264, or 333. Such a second polypeptide may bind to a complementary VH-CH1 polypeptide contained in the fusion protein, for example, in SEQ ID NO: 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 embodiments, the fusion polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO: 362, 363, 325, 286, 579, 581 or 582, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263, 264 or 333. The fusion polypeptides disclosed as SEQ ID NOs: 362, 363, 325, 286, 579, 581 or 582 may be referred to 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 referred to herein as WW0523 (ACP381), WW0524 (ACP382) or WW0556 (ACP414).

[0144] For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 362, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 363, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 325, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO: 286, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO: 264.For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:286, and the second polypeptide may comprise or consist of the amino acid sequence of SEQ ID NO:333. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO:263. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO:264. For example, the fusion protein may comprise or consist of the amino acid sequence of SEQ ID NO:579, and the second polypeptide chain may comprise or consist of the amino acid sequence of SEQ ID NO:233. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:263. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:264. For example, the fusion protein may comprise or consist of SEQ ID NO:581, and the second polypeptide chain may comprise or consist of SEQ ID NO:333. For example, the fusion protein may comprise or consist of SEQ ID NO:582, and the second polypeptide chain may comprise or consist of SEQ ID NO:263. For example, the fusion protein may comprise or consist of SEQ ID NO:582, and the second polypeptide chain may comprise or consist of SEQ ID NO:264. For example, the fusion protein may comprise or consist of SEQ ID NO:582, and the second polypeptide chain may comprise or consist of SEQ ID NO:333.

[0145] Therapeutic methods and pharmaceutical compositions The present disclosure also relates to pharmaceutical compositions comprising one or more fusion proteins disclosed herein, optionally in combination with another therapeutic agent, preferably an immunomodulatory agent or an anti-cancer agent. The present disclosure also relates to the use of such pharmaceutical compositions and the use of one or more fusion proteins, optionally in combination with another therapeutic agent, in the treatment of cancer.

[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. Two or more fusion proteins may be used to provide therapy. For example, the 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 a fusion protein containing an IFN polypeptide.

[0147] The therapeutic combination disclosed herein may comprise a fusion polypeptide cytokine polypeptide [A], a blocking moiety [D], an optional half-life extending moiety [H], and a protease-cleavable polypeptide linker, wherein the cytokine polypeptide, the blocking moiety, and, if present, the optional half-life extending moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated cytokine receptor activating activity, wherein the cytokine receptor activating activity of the fusion polypeptide is at least about 10-fold lower than the cytokine receptor activating activity of a polypeptide containing the cytokine polypeptide generated by cleavage of the protease-cleavable linker, and wherein the fusion polypeptide has a structure of 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); wherein [A] is a cytokine polypeptide, [D] is a blocking moiety, [H] is a half-life extending moiety, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, 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], a blocking moiety [D], an optional half-life extending element [H], and a protease-cleavable polypeptide linker [L], wherein the cytokine polypeptide, cytokine blocking moiety, and, if present, the optional half-life extending element are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated cytokine receptor activating activity, the cytokine receptor activating activity of the fusion polypeptide being at least about 10-fold less than the cytokine receptor activating activity of a polypeptide containing the cytokine polypeptide produced 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); wherein [A] is a cytokine polypeptide, [D] is a blocking moiety, [H] is a half-life extending moiety, [L1] is a protease-cleavable polypeptide linker, [L2] is a polypeptide linker that is optionally protease-cleavable, and [L2'] is a protease-cleavable polypeptide linker. may have:

[0148] The therapeutic drug combination disclosed herein comprises a first fusion protein 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 comprise a fusion protein having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 257 to 300, 302 to 317, 325 to 353, 355 to 365, 366, 372 to 381, 383 to 385, 388 to 420, 579 to 608, 636 to 646, 368 to 371, 434 to 440, 453 to 519, 523 to 538, 421 to 430, and 539 to 578. It is preferable that the first fusion protein and the second fusion protein are different from each other. In some preferred embodiments, the therapeutic drug combination comprises a first 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, and 636-646, and a second fusion protein comprising 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 drug 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 drug 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 drug combinations disclosed herein can include a first fusion protein covalently or non-covalently linked to a second polypeptide chain and a therapeutic agent. The therapeutic combination may comprise (i) a fusion polypeptide comprising an amino acid sequence selected from SEQ ID NO: 362, 363, 325, 286, 579, 581, or 582, and a second polypeptide chain comprising the amino acid sequence of SEQ ID NO: 263, 264, or 333, and (ii) a second therapeutic agent, wherein the second therapeutic agent is 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 a combination thereof. Preferably, 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, chemotherapeutic agents (e.g., Adriamycin, Cervidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Novantrone, Thioguanine, 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 therapy (e.g., CAR-T, T-cell therapy), oncolytic viruses, radiation therapy, and / or small molecules.

[0152] Non-limiting examples of anti-cancer drugs that may be used include acivicin, aclarubicin, acodazole hydrochloride, acronine, adzelesin, aldesleukin, altretamine, ambomycin, amethanthrone acetate, aminoglutethimide, amsacrine, anastrozole, anthramycin, asparaginase, asperlin, azacytidine, azetepa, azotomycin, batimastat, benzodepa, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, bizelesin, bleoma Isin sulfate, brequinar sodium, bropirimine, busulfan, cactinomycin, calsterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzelesin, cedefingol, chlorambucil, ciloremycin, cisplatin, cladribine, crisnatol mesylate, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubicin hydrochloride, decitabine, dexorumaplatin, dezaguanine, dezaguanidin mesylate , diaziquione, docetaxel, doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, elbrozole, esorubicin hydrochloride, estramustine, estramustine sodium phosphate, etanidazole, etoposide, etoposide phosphate, etopurine, Fadrozole hydrochloride, fazarabine, fenretinide, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine, fosquidone, fostriecin sodium salt, gemcitabine, gemcitabine hydrochloride, hydroxyurea, idarubicin hydrochloride, ifosfamide, ilmofosine, 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, liarozole hydrochloride, lometrexol sodium salt, lomustine, losoxantrone hydrochloride, masoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaril, mercaptopurine, methotrexate, methotrexate sodium salt, metoprine, meturedepa, mitindo Mido, mitocalcin, mitochromin, mitogillin, mitomarcine, mitomycin, mitospel, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nocodazole, nogalamycin, ormaplatin, oxisuran, paclitaxel, pegaspargase, periomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobroman, piposulfan, piroxantrone hydrochloride, plicamycin, promestane, porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride, puromycin Isin, puromycin hydrochloride, pyrazofurin, ribopurin, rogletimide, safingol, safingol hydrochloride, semustine, simtrazene, sparfosate sodium salt, sparsomycin, spirogermanium hydrochloride, spiromustine, spiroplatin, streptonigrin, streptozocin, sulofenur, tallysomycin, tecogalan sodium salt, tegafur, teloxantrone hydrochloride, temoporfin, teniposide, teroxilon, testolactone, thiamiprine, thioguanine, thiotepa, tiazofurin, tirapaza Examples of antihistamines include vincristine, toremifene citrate, trestrone acetate, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubrozole hydrochloride, uracil mustard, uredepa, vapreotide, verteporfin, vinblastine sulfate, vincristine sulfate, vindesine, vindestine sulfate, vinepidine sulfate, vinglisinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinzolidine sulfate, vinzolidine sulfate, vorozole, zeniplatin, zinostatin, and zorubicin hydrochloride.

[0153] Accordingly, the present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with a chemotherapeutic agent, such as adriamycin, cerbidine, bleomycin, alkeran, velban, oncovin, fluorouracil, thiotepa, methotrexate, bisantrene, noantrone, thiguanine, cytarabine, or procarbazine. The present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with an antibody-drug conjugate. Various antibody-drug conjugates suitable for use in cancer therapy are well known and typically include an antibody that binds to a cellular antigen preferentially expressed or expressed at high levels on tumor cells, and a cytotoxic drug. The present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with cell therapy, e.g., CAR-T or T-cell therapy. The present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with an oncolytic virus. Exemplary oncolytic viruses include oncolytic adenovirus, herpes simplex virus type 1 (HSV), poliovirus, measles virus (MV), Newcastle disease virus (NDV), reovirus, vesicular stomatitis virus (VSV), and Zika virus. The present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with radiation therapy, e.g., external beam radiation therapy or internal therapeutic radiation therapy.

[0154] The present disclosure relates to therapeutic combinations of any of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) in combination with a cytokine (e.g., IL-2, IL-15), a signal transduction inhibitor (e.g., a BRAF inhibitor or a MEK inhibitor), a checkpoint inhibitor (e.g., PDL-1, PD-1, CTLA-4), a c-met inhibitor, a kinase inhibitor (e.g., a VEGF inhibitor), a proteasome inhibitor, an mTOR inhibitor, or an angiogenesis inhibitor. In embodiments, any one of the fusion proteins disclosed herein (e.g., a fusion protein comprising an IL-2 polypeptide and an IL-12 polypeptide or an IFN polypeptide) may be combined with an anti-PD-L1 agent or an anti-PD-1 agent. Exemplary PD-1 and / or PD-L1 inhibitors include, but are not limited to, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, dostallimab, INCMGA00012, AMP-224, and AMP-514. In embodiments, a 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, 421-430, and 539-578 may be combined with a checkpoint inhibitor, such as PDL-1, PD-1, or CTL-4.

[0155] Further provided are methods for treating a subject having or at risk of developing a disease or disorder, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immunological disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The methods disclosed herein are preferably used to treat a subject with cancer. The methods involve administering to a subject in need thereof an effective amount of a fusion protein disclosed herein, typically administered as a pharmaceutical composition. In some embodiments, the method further comprises selecting a subject having or at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises a blocked cytokine activated at a site of inflammation or a tumor, or a fragment, variant, subunit, or mutein thereof. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment, or mutein thereof, and a serum half-life extending element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a variant, subunit, fragment, or mutein thereof, and a blocking moiety, such as a steric blocking polypeptide, which can sterically block the action of the cytokine polypeptide, fragment, or mutein thereof. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutein thereof, a blocking moiety, and a serum half-life extending element.

[0156] Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. It is a defensive response involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cellular damage, remove damaged necrotic cells and tissue from the initial injury and inflammatory process, and initiate tissue repair. Inflammation can arise from infection, as a symptom, or as a disease, such as cancer, atherosclerosis, allergies, myopathy, HIV, obesity, or autoimmune disease. Autoimmune diseases are chronic conditions resulting from an abnormal immune response to self-antigens. Autoimmune diseases that can be treated by the 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 include one or more protease-cleavable linker sequences. The linker sequence serves to provide flexibility between polypeptides so that each polypeptide can inhibit the action of the first polypeptide. The linker sequence may be located between any or all of the cytokine polypeptide, fragment or mutein thereof, blocking moiety, and serum half-life extending element. Optionally, the composition includes two, three, four, or five linker sequences. The linker sequence, 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 protease.

[0159] Suitable linkers may vary in length, for example, from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 to 15 amino acids, from 3 to 12 amino acids, for example, from 4 to 10 amino acids, from 1 to 9 amino acids, from 6 to 8 amino acids, or from 7 to 8 amino acids, and may 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] Also provided are methods for treating a subject having or at risk of developing cancer. The methods include administering to a subject in need thereof an effective amount of a chimeric polypeptide (fusion protein) described herein, typically administered as a pharmaceutical composition. In some embodiments, the methods further include selecting a subject having or at risk of developing cancer. The pharmaceutical composition preferably includes a blocked cytokine, fragment, or mutein thereof, that is activated at the tumor site.

[0161] The methods disclosed herein can be used to treat any suitable cancer, including hematopoietic malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Exemplary suitable cancers include acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary, cardiac tumor, cervical cancer, chondroma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, embryonal carcinoma, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye 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's 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, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma Ranoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary site, 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 carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid carcinoma, penile cancer, pharyngeal cancer, chromaffin These include germ cell tumors, pituitary tumors, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, kidney cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumors, gastric cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor.

[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, Cervidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Novantrone, Thioguanine, Cytaribine, Procarabizine), immuno-oncology agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2, VEGF inhibitors), cell therapy (e.g., CAR-T, T-cell therapy), oncolytic viruses, radiation therapy, etc.

[0164] In embodiments, the fusion proteins described herein can be administered with one or more additional inducible cytokine fusion proteins. For example, a fusion protein containing an IL-2 polypeptide as described herein can be administered 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 can be administered 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 can be administered 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 comprising the amino acid sequence of any one 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 comprising the amino acid sequence of any one 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 comprising the amino acid sequence of any one of SEQ ID NOs: 368-371, 434-440, 453-519, or 523-538 may be administered together with a second, different fusion protein comprising the amino acid sequence of any one 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 comprising the amino acid sequence of any one of SEQ ID NOs: 421-430 and 539-578 may be administered together with a second, different fusion protein comprising the amino acid sequence of any one 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 a combination thereof.

[0166] Additional 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 induction 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., VEGF inhibitors), proteasome inhibitors, mTOR inhibitors, and angiogenesis inhibitors.

[0167] Preferred immuno-oncology agents are anti-PD-L1 agents or anti-PD-1. Exemplary PD-1 and / or PD-L1 inhibitors include, but are not limited to, spartalizumab, camrelizumab, sintilimab, tislelizumab, toripalimab, 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 a checkpoint inhibitor, such as PDL-1, PD-1, or CTL-4.

[0168] Disclosed herein is a pharmaceutical formulation or composition 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 refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject without causing undesired biological effects or interacting in a deleterious manner with other components of the pharmaceutical formulation or composition containing it. The carrier is selected to minimize 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 stEdition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation isotonic, although the formulation may also 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 preparations, such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptide. The matrices are in the form of shaped articles, e.g., membranes, liposomes, or microparticles. Certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered. The carrier is suitable for administering the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide to humans or other subjects.

[0170] Pharmaceutical formulations or compositions are administered in several ways, depending on whether local or systemic treatment is desired and the area to be treated. The compositions are administered by any of several modes of administration, including topical, oral, parenteral, intravenous, intraarticular, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, intrahepatic, intracranial, nebulized / inhaled, or via placement through a bronchoscope. In some embodiments, the compositions are administered locally (non-systemically), including intratumorally, intraarticularly, intrathecally, 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 saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (e.g., those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases and the like, are optionally present.

[0172] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are optionally necessary or desirable.

[0173] Compositions for oral administration include powders or granules, suspensions or solutions made in water or non-aqueous media, capsules, pre-packaged tablets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0174] Optionally, the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide is administered via a vector. There are several compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells either in vitro or in vivo, for example, via expression vectors. These methods and compositions can be broadly 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 to a subject. The components of the chimeric nucleic acids disclosed herein are typically operably linked in-frame to encode a fusion protein.

[0175] As used herein, a plasmid or viral vector is an agent that transports the nucleic acids of the present disclosure into cells without degradation and contains a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the recipient cell. Viral vectors include, for example, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, poliovirus, Sindbis, and other RNA viruses, such as those with an HIV backbone. Also preferred are any viral families that share the properties that make them suitable for use as vectors. Retroviral vectors are generally described 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. Construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., J. Virol. 61:1213-20 (1987)). (e.g., Haj-Ahmad 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 utility of these viruses as vectors is that they are limited in the extent to which they can spread to other cell types because, although they can replicate within the initially infected cell, they are unable to form new infectious viral particles. Recombinant adenoviruses have been shown to achieve high efficiency following direct in vivo delivery to respiratory epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and multiple other tissue sites. Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.

[0176] The provided polypeptides and / or nucleic acid molecules can be delivered by virus-like particles. Virus-like particles (VLPs) consist of viral protein(s) derived from viral structural proteins. Methods for making and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0177] The provided polypeptides can be delivered by subviral dense bodies (DBs), which transport proteins into target cells by membrane fusion. Methods for making and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0178] The provided polypeptides can be delivered by tegument aggregates. Methods for making and using tegument aggregates are described in International Publication No. WO2006 / 110728.

[0179] Non-viral delivery methods can include expression vectors containing a nucleic acid molecule and a nucleic acid sequence encoding the polypeptide, where the nucleic acid is operably linked to an expression control sequence. Suitable vector backbones include, for example, those commonly 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. 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 initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to generate chimeric polypeptides by expression in suitable host cells, such as CHO cells.

[0180] Preferred promoters controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, for example, from the genomes of viruses such as polyoma, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus (CMV), or from heterologous mammalian promoters, such as the β-actin promoter or EF1α promoter, or hybrid or chimeric promoters (e.g., the CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0181] Enhancers generally refer to DNA sequences that function at variable distances from the transcription start site and can be located either 5' or 3' of the transcription unit. Furthermore, enhancers can be found within introns or within the coding sequence itself. They are usually 10 to 300 base pairs (bp) in length and function in cis. Enhancers generally function to increase transcription from nearby promoters. Enhancers may also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), enhancers from eukaryotic viruses will typically be used for normal expression. Preferred examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0182] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated, for example, by environmental factors, such as temperature and light. Optionally, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In some vectors, the promoter and / or enhancer region may be active in a cell type-specific manner. Optionally, in some vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and retroviral long terminal repeats (LTRs).

[0183] A vector may also contain, for example, an origin of replication and / or a marker. A marker gene may confer a selectable phenotype, such as antibiotic resistance, to a cell. The marker product is used to determine whether the vector has been delivered to a cell and, once delivered, whether it is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. Such selectable markers can be successfully transferred into mammalian host cells, allowing the transformed mammalian host cells to survive when placed under selection pressure. Other examples of markers include, for example, E. coli selectable markers. Examples of such tags include the lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, expression vectors can contain tag sequences designed to facilitate 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.), are typically expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within 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 refer to two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide 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 comprising the molecule, and that the peptides of the present invention may contain fewer or more amino acid residues. The term "subject," as used throughout, may be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, or any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be encompassed. As used herein, the terms "patient" or "subject" may be used interchangeably and may refer to a subject with a disease or disorder (e.g., cancer). The terms "patient" or "subject" include human and veterinary subjects.

[0185] A subject at risk for developing a disease or disorder may be genetically predisposed to the disease or disorder, for example, may have a family history, or may have a mutation in a gene that causes the disease or disorder, or may show early signs or symptoms of the disease or disorder. A subject who currently has a disease or disorder may have one or more symptoms of the disease or disorder and may have been diagnosed with the disease or disorder.

[0186] The methods and medicaments described herein are useful for both prophylactic and therapeutic treatments. For prophylactic use, a therapeutically effective amount of a chimeric polypeptide or a chimeric nucleic acid sequence encoding a chimeric polypeptide described herein is administered to a subject pre-onset (e.g., before overt signs of cancer or inflammation) or during early onset (e.g., at the onset of early signs and symptoms of cancer or inflammation). Prophylactic administration can occur from days to years before symptoms of cancer or inflammation become apparent. Prophylactic administration can be used, for example, in the prophylactic treatment of subjects diagnosed with a genetic predisposition to cancer. Therapeutic treatment includes administering a therapeutically effective amount of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide described herein to a subject after the diagnosis or development of cancer or inflammation (e.g., an autoimmune disease). Prophylactic use can also be applied when a patient is undergoing a treatment in which inflammation is expected, such as chemotherapy.

[0187] According to the methods taught herein, a subject is administered an effective amount of an agent (e.g., a chimeric polypeptide). The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount required to produce a desired physiological response. The effective amount and the regimen for administering an agent can be determined empirically, and making such determinations is within the skill of the art. The dosage range for administration is one large enough to produce the desired effect in which one or more symptoms of a disease or disorder are affected (e.g., alleviated or delayed). The dosage should not be so large as to cause significant adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Typically, dosages vary 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 regimen, and can be determined by one of ordinary skill in the art. Dosages can be adjusted by the individual physician in the event of any contraindications. Dosages can vary and can be administered in one or more doses daily for one or several days. Guidance as to appropriate dosages for a given class of pharmaceutical product can be found in the literature.

[0188] As used herein, the terms treatment, treating, or treating refer to a method of reducing the effects of a disease or condition, or the symptoms of a disease or condition. Thus, in the methods of the present disclosure, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction 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 therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to the original 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 prevent, preventing, and prevention of a disease or disorder refer to an action that suppresses or delays the onset or progression of one or more symptoms of the disease or disorder, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, occurring before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder. As used herein, references to decreasing, reducing, or inhibiting include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to control levels. Such terms can, but do not necessarily, include complete elimination.

[0190] IL-2 variants have been developed that are selective for IL2Rαβγ over IL2Rβγ (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. Because IL-2 has undetectable affinity for IL2RG, these variants consequently have reduced affinity for the IL2Rβγ receptor complex and a reduced ability to activate IL2Rβγ-expressing cells, while retaining the 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), has been clinically tested as a less toxic form of IL-2 as an immune system stimulator, based on the hypothesis that IL2Rβγ-expressing NK cells are the primary cause of toxicity. Bay 50-4798 has been shown to selectively stimulate the proliferation of activated T cells versus NK cells, and has been shown to be effective in cancer patients (Margolin, K., et al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, R.T., et al., 2008, J. Bay 50-4798 was evaluated in phase I / II trials in the US Interferon Cytokine Res., 28:89-100. These trials demonstrated that Bay 50-4798 was significantly safer and better tolerated than aldesleukin and also showed that it increased levels of CD4+CD25+ T cells, a population enriched for Treg cells. Subsequent to these trials, work in the field has more fully established the identity of Treg cells and demonstrated that Treg cells selectively express IL2Rαβγ (reviewed in Malek, TR, et al., 2010, Immunity, 33:153-65).

[0192] In addition, variants can be made that selectively alter affinity for the CD25 chain relative to native Il-2.

[0193] IL-2 can be engineered to generate mutants that bind to the IL-2R complex overall or specifically to the IL-2R α subunit with different affinities than the corresponding wild-type IL-2 or a currently available mutant (designated C125S because the cysteine ​​residue at position...

Claims

[Claim 1] The invention described in the specification.

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