Il-2 compositions and methods of use thereof

Abstract

To provide IL-2 compositions, and methods of use thereof.SOLUTION: Provided are activatable proprotein homodimers comprising at least two separate polypeptide chains, in which each chain comprises an IL-2 protein, a cleavable linker, and an IL-2 binding protein, among some optional features, as well as related pharmaceutical compositions and methods of use thereof are provided. Interleukin-2 (IL-2) immunotherapy has proven useful in the treatment of cancers, such as malignant melanoma and renal cell carcinoma, and chronic infections, such as HIV infection.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 908,782, filed October 1, 2019, and U.S. Provisional Patent Application No. 62 / 873,399, filed July 12, 2019, each of which is incorporated by reference in its entirety herein.

[0002] Description of sequence listing The sequence listing associated with this application is provided in text format in lieu of a paper copy and is hereby incorporated by reference. The name of the text file containing the sequence listing is PRVA_003_02WO_ST25.txt. This text file is 952KB, was created on July 9, 2020, and has been submitted electronically via EFS-Web.

[0003] background Technical Field The present disclosure relates to activatable proprotein homodimers comprising at least two separate polypeptide chains, each chain comprising an IL-2 protein, a cleavable linker, and an IL-2 binding protein, among other optional features, and related pharmaceutical compositions and methods of use thereof. [Background technology]

[0004] 2. Description of Related Art Interleukin-2 (IL-2) immunotherapy has proven useful in the treatment of cancers such as malignant melanoma and renal cell carcinoma, as well as chronic infectious diseases such as HIV infection.

[0005] However, there are certain problems associated with most IL-2 therapies. For example, current forms of IL-2 therapy have a short half-life in the circulation and primarily inhibit the immune-suppressing regulatory T cells, or T reg(See, e.g., Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015). Similarly, the effects of IL-2 treatment are primarily systemic rather than localized to target tissues, resulting in many severe side effects, such as breathing problems, nausea, low blood pressure, loss of appetite, confusion, severe infections, seizures, allergic reactions, cardiac problems, renal failure, and vascular leak syndrome. Nonetheless, IL-2 treatment can be effective, and there is an unmet need in the art to overcome these and other shortcomings. Embodiments of the present disclosure address these problems and more by providing activatable proproteins, including IL-2, that can be activated in diseased tissue, such as cancerous tissue or tumors. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015 Summary of the Invention [Means for solving the problem]

[0007] Quick Overview An embodiment of the present disclosure is an activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, (a) the first polypeptide and the second polypeptide comprise, in N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein; or (b) the first polypeptide and the second polypeptide comprise, in N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein; a binding moiety of a first polypeptide binds to a binding moiety of a second polypeptide, an IL-2 protein of the first polypeptide binds to an IL-2 binding protein of the second polypeptide, and the IL-2 binding protein of the first polypeptide binds to an IL-2 protein of the second polypeptide, said (overall) binding masking a binding site of an IL-2 protein(s) that would otherwise bind to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell in vitro or in vivo, and at least one of the first or second linkers is a cleavable linker; or (c) the first and second polypeptides comprise, in an N-to-C-terminal or C-to-N-terminal direction, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and an affinity purification tag; or (d) the first and second polypeptides comprise, in an N-to-C-terminal or C-to-N-terminal direction, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and an affinity purification tag; The IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and the IL-2 binding protein of the first polypeptide binds to the IL-2 protein of the second polypeptide, and said (total) binding is in vitro or in vivo. It comprises an activatable proprotein homodimer that masks the binding site of an IL-2 protein(s) that would otherwise bind to the IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vivo, and in which the first linker is a cleavable linker.

[0008] In some embodiments, the first and second IL-2 proteins comprise, consist, or consist essentially of an amino acid sequence selected from Table S1, optionally an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 21-153 of SEQ ID NO:1 (full length wild type human IL-2), optionally comprising a C145X (wherein X is any amino acid) or C145S substitution as defined by SEQ ID NO:1. In some embodiments, the first and second IL-2 proteins comprise, consist, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:2 (mature human IL-2 with a C125S substitution), optionally wherein the IL-2 protein retains the S125 residue as defined by SEQ ID NO:2. In some embodiments, the first and second IL-2 proteins comprise one or more substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined by SEQ ID NO:2.

[0009] In some embodiments, the first IL-2 protein forms a disulfide bond with the second IL-2 binding protein, and the second IL-2 protein forms a disulfide bond with the first IL-2 binding protein, optionally via one or more of the cysteines described in claim 4 and one or more cysteines in the first and second IL-2 binding protein(s).

[0010] In some embodiments, the first and second IL-2 proteins comprise one or more amino acid substitutions at positions 69, 74, and / or 128 as defined by SEQ ID NO:2, optionally wherein the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO:2. In some embodiments, the first and second IL-2 proteins comprise one or more amino acid substitutions at positions T3, R38, F42, Y45, E61, E62, E68, and / or L72 as defined by SEQ ID NO:2, optionally including combinations thereof: T3A; R38A and R38K; F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, and F42I; Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K; E61S; E62A and E 62L; E68A and E68V; and combinations selected from L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, optionally F42A, Y45A, and L72G; R38K, F42Q, Y45N, E62L, and E68V; R38K, F42Q, Y45E, and E68V; R38A, F42I, Y45N, E62L, and E68V; R38K, F42K, Y45R, E62L, and E68V; R38K, F42I, Y45E, and E68V; and R38A, F42A, Y45A, and E62A.

[0011] In some embodiments, the first and second IL-2 proteins comprise, consist, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:3 (mature human IL-2 "D10" variant), optionally wherein the IL-2 protein retains any one or more of the Q74H, L80F, R81D, L85V, I86V, and / or I92F substitutions defined by SEQ ID NO:3.

[0012] In some embodiments, the first and second IL-2 binding proteins comprise a first and second IL-2Rα protein, or a first and second antibody or antigen-binding fragment thereof that specifically binds to an IL-2 protein(s), optionally a bispecific antibody or antigen-binding fragment thereof.

[0013] In some embodiments, the first and second IL-2Rα proteins comprise, consist of, or consist essentially of an amino acid sequence selected from Table S2, optionally an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 22-187 of SEQ ID NO:4 (full length wild-type human IL-2Rα). In some embodiments, the first and second IL-2Rα proteins comprise one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C, and / or a K38S substitution as defined by SEQ ID NO:6 (human IL-2Rα Sushi1-Sushi2 domains). In some embodiments, optionally one or more of the cysteines of claim 11, and one or more cysteines in the IL-2 protein, optionally one or more of the cysteines of claim 4, optionally IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, IL2-R38C and IL2Rα-H120C, IL2-T41C and IL2Rα-I118C, IL2-F42C and IL2Rα-N27C, IL2-T42C and IL2Rα-T27C, IL2-T41C and IL2Rα-T118C, IL2-T42C and IL2Rα-T118C, IL2-T42C and IL2Rα-T27C, IL2-T42C and IL2Rα-T118C, IL2-T42C and IL2Rα-T27C, IL2-T42C and IL2Rα-T42 ... The first IL-2Rα protein forms a disulfide bond with the second IL-2 protein, and the second IL-2Rα protein forms a disulfide bond with the first IL-2 protein, via one or more cysteine ​​pairs selected from L2-E61C and IL2Rα-K38C, IL2-E61C and IL2Rα-S39C, and IL2-V69C and IL2Rα-L42C, and the disulfide bond between the IL-2 protein and the IL-2Rα protein is regIn some embodiments, the first and second IL-2Rα proteins contain alanine substitutions at positions 49 and / or 68 as defined by SEQ ID NO:6.

[0014] In some embodiments, the first and second antibodies or antigen-binding fragments thereof that specifically bind to IL-2 protein are selected from one or more of a whole antibody, a Fab, a Fab', a F(ab')2, a monospecific Fab2, a bispecific Fab2, a FV, a single chain Fv (scFv), a scFV-Fc, a nanobody, a diabody, a camelid antibody, and a minibody, and optionally the antibody is NARA1 or an antigen-binding fragment thereof. In some embodiments, the binding moieties of (a) and / or (b) do not bind to IL-2 protein or to an IL-2 binding protein. In some embodiments, the binding moieties of (a) and / or (b) bind to IL-2 protein. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) bind to each other through at least one non-covalent interaction, and optionally form a homodimer. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) bind to each other through at least one covalent bond, and optionally form a homodimer. In some embodiments, the at least one covalent bond comprises at least one disulfide bond.

[0015] In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) are selected from Table M1. In some embodiments, the binding moieties of the first and second polypeptides of (a) or (b) comprise an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) comprise a CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or a CL domain of an immunoglobulin, including fragments and variants thereof. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) comprise, from the N- to C-terminus: (1) an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof; and (2) a CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or a CL domain of an immunoglobulin, including fragments and variants thereof. In some embodiments, the antigen-binding domain comprises an immunoglobulin VH or VL domain, including antigen-binding fragments and variants thereof. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) do not bind to an antigen. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) comprise an immunoglobulin CH2CH3 domain. In some embodiments, the immunoglobulin is from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. In some embodiments, the binding moieties of the first and second polypeptides of (a) and / or (b) comprise a leucine zipper peptide.

[0016] In some embodiments, the affinity purification tag of (c) and / or (d) is selected from a polyhistidine tag (optionally a hexahistidine tag), a VSV-G tag, a universal tag, a Strep tag, an S tag, an S1 tag, a Phe tag, a Cys tag, an Asp tag, an Arg tag, a Myc epitope tag, a KT3 epitope tag, an HSV epitope tag, a histidine affinity tag, a hemagglutinin (HA) tag, a FLAG epitope tag, an E2 epitope tag, a V5 tag, a T7 tag, an AU5 epitope tag, and an AU1 epitope tag.

[0017] In some embodiments, the cleavable linker comprises a protease cleavage site, and optionally the cleavable linker is selected from Table S3. In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of metalloproteases, serine proteases, cysteine ​​proteases, and aspartic acid proteases. In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A, and cathepsin B. In some embodiments, the first linker and / or the second linker is about 1-50, about 1-40, about 1-30, about 1-20, about 1-10, about 1-5, about 1-4, about 1-3 amino acids in length, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16 , about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 amino acids in length.

[0018] In some embodiments, the first linker (a) and / or (b) is a cleavable linker and the second linker (a) and / or (b) is a non-cleavable linker. In some embodiments, cleavage of the first linker (a) and / or (b), optionally by a protease, exposes a binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo. In some embodiments, the first linker (a) and / or (b) is a non-cleavable linker and the second linker (a) and / or (b) is a cleavable linker. In some embodiments, cleavage of the second linker (a) and / or (b), optionally by a protease, exposes a binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo. In some embodiments, cleavage of the first linker of (c) and / or (d), optionally by a protease, exposes a binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo. In some embodiments, the immune cell is selected from one or more of a T cell, a B cell, a natural killer cell, a monocyte, and a macrophage.

[0019] In some embodiments, the first and second polypeptides of (a) comprise, from N to C terminal, a binding moiety, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein. In some embodiments, the first and second polypeptides of (a) comprise, from N to C terminal, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and a binding moiety. In some embodiments, the first and second polypeptides of (b) comprise, from N to C terminal, a binding moiety, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein. In some embodiments, the first and second polypeptides of (b) comprise, from N to C terminal, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and a binding moiety. In some embodiments, the first and second polypeptides of (c) comprise, from N-to-C-terminus, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and an affinity purification tag. In some embodiments, the first and second polypeptides of (d) comprise, from N-to-C-terminus, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and an affinity purification tag.

[0020] In some embodiments, the first polypeptide and the second polypeptide comprise, consist, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a sequence selected from Table S4, optionally with a TEV protease cleavage site replaced with a cleavage site cleavable by a human protease, optionally with a cleavable linker selected from Table S3.

[0021] In some embodiments, the activatable proprotein is in a substantially homodimeric form in physiological solution or under physiological conditions, optionally under in vivo conditions.

[0022] Also included are recombinant nucleic acid molecules encoding the activatable proprotein homodimers described herein, vectors comprising the recombinant nucleic acid molecules described herein, and host cells comprising the recombinant nucleic acid molecules or vectors described herein.

[0023] Also included is a method of producing an activatable proprotein, comprising culturing a host cell described herein under culture conditions suitable for expression of an activatable proprotein homodimer, and isolating the activatable proprotein from the culture.

[0024] Also included is a pharmaceutical composition comprising an activatable proprotein homodimer as described herein and a pharma- ceutically acceptable carrier.

[0025] Certain embodiments include a method of treating a disease in a subject and / or a method of enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein.

[0026] In some embodiments, the disease is selected from one or more of a cancer, a viral infection, and an immune disorder. In some embodiments, the cancer is a primary or metastatic cancer and is selected from one or more of melanoma (optionally metastatic melanoma), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0027] In some embodiments, after administration, the activatable proprotein homodimer is activated through cleavage by a protease in a cell or tissue, optionally a cancer cell or tissue, to expose a binding site(s) for a first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo, thereby generating an activated protein. In some embodiments, the activated protein binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo via an IL-2 protein. In some embodiments, the immune cell is selected from one or more of a T cell, a B cell, a natural killer cell, a monocyte, and a macrophage. In some embodiments, the bond between the IL-2 protein(s) and the IL-2 binding protein(s) in the activated protein (optionally a disulfide bond between the IL-2 protein(s) and the IL-2Rα protein(s)) is activated by a T cell. reg IL-2Rα / β / γc chains expressed on T reg This interferes with the binding of the activator protein to the

[0028] In some embodiments, administration and activation of an activatable proprotein enhances the immune response in a subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, optionally wherein the immune response is an anti-cancer or anti-viral immune response. In some embodiments, administration and activation of an activatable proprotein increases cell killing in a subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, or more than about 100% compared to a control. %, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, and optionally the cell killing is the killing of cancer cells or the killing of virally infected cells.

[0029] In some embodiments, the viral infection is selected from one or more of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, hepatitis E, calicivirus-associated diarrhea, rotavirus diarrhea, Haemophilus influenzae type B pneumonia and invasive disease, influenza, measles, mumps, rubella, parainfluenza-associated pneumonia, respiratory syncytial virus (RSV) pneumonia, severe acute respiratory syndrome (SARS), human papillomavirus, herpes simplex type 2 genital ulcers, dengue fever, Japanese encephalitis, tick-borne encephalitis, West Nile virus-associated disease, yellow fever, Epstein-Barr virus, Lassa fever, Crimean-Congo hemorrhagic fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, rabies, Rift Valley fever, smallpox, upper and lower respiratory tract infections, and poliomyelitis, and optionally, the subject is HIV positive.

[0030] In some embodiments, the immune disorder is selected from one or more of type I diabetes, vasculitis, and immunodeficiency.

[0031] In some embodiments, the pharmaceutical composition is administered to the subject by parenteral administration. In some embodiments, the parenteral administration is intravenous administration.

[0032] Also included is the use of a pharmaceutical composition as described herein in the preparation of a medicament for treating a disease in a subject and / or enhancing an immune response in a subject. Certain embodiments include a pharmaceutical composition as described herein for use in treating a disease in a subject and / or enhancing an immune response in a subject. [Brief description of the drawings]

[0033] [Figure 1A] FIG. 1A shows the protein topology of human interleukin 2 (IL-2) and the human interleukin 2 receptor alpha chain (IL-2Rα). [Figure 1B]FIG. 1B shows the quaternary structure of IL-2 complexed with its receptors IL-2Rα (CD25), IL-2Rβ (CD122), and the common gamma chain (CD132) (PDB: 2ERJ). [Figure 2-1] FIG. 2A illustrates the fusion of the C-terminus of IL-2 to the N-terminus of IL-2Rα via a cleavable / non-cleavable linker. An optional His tag is added to the C-terminus of IL-2Rα to facilitate purification. A schematic homodimeric structure is presented. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. IL-2 activity can be restored after cleavage by a protease between IL-2 and IL-2Rα. FIG. 2B illustrates a protein sequence motif and conformation diagram for the protein described in FIG. 2A. [Figure 2-2] FIG. 2C illustrates the fusion of the C-terminus of Fc to the N-terminus of IL-2 with a cleavable / non-cleavable linker, and the fusion of the C-terminus of IL-2 to the N-terminus of IL-2Rα with a cleavable / non-cleavable linker. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. Partial activity can be restored after protease cleavage between Fc and IL-2, and full activity can be restored after protease cleavage between IL-2 and IL-2Rα, or between IL-2 / IL-2Rα and Fc / IL-2. FIG. 2D illustrates protein sequence motifs and configuration diagrams for the proteins described in FIG. 2C. [Figure 2-3]FIG. 2E illustrates the fusion of the C-terminus of IL-2 to the N-terminus of IL-2Rα via a cleavable / non-cleavable linker, and the C-terminus of IL-2Rα to the N-terminus of Fc via a cleavable / non-cleavable linker. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. Partial activity can be restored after protease cleavage between Fc and IL-2, and full activity can be restored after protease cleavage between IL-2 and IL-2Rα or between IL-2 / IL-2Rα and Fc / IL-2Rα. FIG. 2F illustrates protein sequence motifs and configuration diagrams for the proteins described in FIG. 2E. [Figure 3-1] FIG. 3A illustrates the fusion of the C-terminus of IL-2Rα to the N-terminus of IL-2 via a cleavable / non-cleavable linker. An optional His tag is added to the C-terminus of IL-2Rα to facilitate purification. The predicted homodimeric structure is presented. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. IL-2 activity can be restored after protease cleavage between IL-2 and IL-2Rα. FIG. 3B illustrates protein sequence motifs and conformation diagrams for the proteins described in FIG. 3A. [Figure 3-2] FIG. 3C illustrates the fusion of the C-terminus of Fc to the N-terminus of IL-2Rα via a cleavable / non-cleavable linker, and the fusion of the C-terminus of IL-2Rα to the N-terminus of IL-2 via a cleavable / non-cleavable linker. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. Partial activity can be restored after protease cleavage between Fc and IL-2, and full activity can be restored after protease cleavage between IL-2 and IL-2Rα or between IL-2 / IL-2Rα and Fc / IL-2Rα. FIG. 3D illustrates protein sequence motifs and configuration diagrams for the proteins described in FIG. 3C. [Figure 3-3] FIG. 3E illustrates the fusion of the C-terminus of IL-2Rα to the N-terminus of IL-2 via a cleavable / non-cleavable linker, and the fusion of the C-terminus of IL-2 to the N-terminus of Fc via a cleavable / non-cleavable linker. IL-2 in this fusion protein cannot bind to and signal through the IL-2Rβ / γc receptor. Partial activity can be restored after protease cleavage between Fc and IL-2, and full activity can be restored after protease cleavage between IL-2 and IL-2Rα or between IL-2 / IL-2Rα and Fc / IL-2. FIG. 3F illustrates protein sequence motifs and configuration diagrams for the proteins described in FIG. 3E. [Figure 4A] FIG. 4A shows a schematic diagram of activation of the “IL-2-linker-IL-2Rα-linker-His6” activatable proprotein through protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα. [Figure 4B] FIG. 4B shows a schematic diagram of activation of the “Fc-linker-IL-2-linker-IL-2Rα” activatable proprotein through protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα. [Figure 4C] FIG. 4C shows a schematic diagram of activation of the “IL-2-linker-IL-2Rα-linker-Fc” activatable proprotein through protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα. [Figure 4D] FIG. 4D shows a schematic diagram of activation of the “IL-2-linker-IL-2Rα-linker-Fc” activatable proprotein through protease cleavage of the substrate linker sequences between IL-2 / IL-2Rα and IL-2Rα / Fc. [Figure 4E] FIG. 4E shows a schematic diagram of partial activation of the “IL-2-linker-IL-2Rα-linker-Fc” activatable proprotein through protease cleavage of the substrate linker sequence between IL-2Rα and Fc. [Figure 5-1]Figure 5A illustrates the fusion of the C-terminus of a binding moiety to the N-terminus of an IL-2 protein by a cleavable / non-cleavable linker, and the fusion of the C-terminus of an IL-2 protein to the N-terminus of an IL-2 binding protein by a cleavable / non-cleavable linker. Figure 5B illustrates the fusion of the C-terminus of an IL-2 protein to the N-terminus of an IL-2 binding protein by a cleavable / non-cleavable linker, and the fusion of the C-terminus of an IL-2 binding protein to the N-terminus of a binding moiety by a cleavable / non-cleavable linker. [Figure 5-2] Figure 5C illustrates fusing the C-terminus of a binding moiety to the N-terminus of an IL-2 binding protein via a cleavable / non-cleavable linker, and fusing the C-terminus of an IL-2 binding protein to the N-terminus of an IL-2 protein via a cleavable / non-cleavable linker. Figure 5D illustrates fusing the C-terminus of an IL-2 binding protein to the N-terminus of an IL-2 protein via a cleavable / non-cleavable linker, and fusing the C-terminus of an IL-2 protein to the N-terminus of a binding moiety via a cleavable / non-cleavable linker. [Figure 6-1] Figures 6A-6C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 6A shows the results of non-reducing SDS-PAGE, 6B shows the results of reducing SDS-PAGE, and 6C shows the results of cleavage. "M" on the figure represents a protein standard marker. On Figure 6C, "1" represents the protein before cleavage by TEV, and "2" represents the protein after cleavage by TEV. [Figure 6-2] Same as above. [Figure 7-1] 7A-7J illustrate representative HPLC analysis results of purified proteins. [Figure 7-2] 7A-7J illustrate representative HPLC analysis results of purified proteins. [Figure 7-3] 7A-7J illustrate representative HPLC analysis results of purified proteins. [Figure 7-4] 7A-7J illustrate representative HPLC analysis results of purified proteins. [Figure 7-5] 7A-7J illustrate representative HPLC analysis results of purified proteins. [Figure 8-1] Figures 8A-8L and Figures 9A-9E illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 8-2] Figures 8A-8L and Figures 9A-9E illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 9] Figures 8A-8L and Figures 9A-9E illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 10] Figures 10A-10C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 10A shows the results of non-reducing SDS-PAGE, 10B shows the results of reducing SDS-PAGE, and 10C shows the results of cleavage. "M" on the figure represents a protein standard marker. On Figure 10C, "1" represents the protein before cleavage by TEV, and "2" represents the protein after cleavage by TEV. [Figure 11-1] 11A-11F illustrate representative HPLC analysis results of purified proteins. [Figure 11-2] 11A-11F illustrate representative HPLC analysis results of purified proteins. [Figure 11-3] 11A-11F illustrate representative HPLC analysis results of purified proteins. [Figure 12] 12A-12F illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 13]Figures 13A-13C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion proteins. 13A shows the results of non-reducing SDS-PAGE, 13B shows the results of reducing SDS-PAGE, and 13C shows the results of cleavage. "M" on the figures represents a protein standard marker. On Figure 13C, "1" represents the protein before cleavage with uPA, and "2" represents the protein after cleavage with uPA. [Figure 14-1] 14A-14D illustrate representative HPLC analysis of the purified protein. [Figure 14-2] 14A-14D illustrate representative HPLC analysis of the purified protein. [Figure 15] 15A-15E illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 16-1] Figures 16A-16C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 16A shows the results of non-reducing SDS-PAGE, 16B shows the results of reducing SDS-PAGE, and 16C shows the results of cleavage. "M" on the figure represents a protein standard marker. On Figure 16C, "1" represents the protein before cleavage by TEV or uPA, and "2" represents the protein after cleavage by TEV or uPA. (P1773-P1778 cleaved by TEV; P1779-P1785 cleaved by uPA). [Figure 16-2] Same as above. [Figure 17-1] 17A-17D illustrate representative HPLC analysis of the purified protein. [Figure 17-2] 17A-17D illustrate representative HPLC analysis of the purified protein. [Figure 18-1] 18A-18N illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 18-2]18A-18N illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 18-3] 18A-18N illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 19-1] Figures 19A-19D show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 19A shows the results of non-reducing SDS-PAGE, 19B shows the results of reducing SDS-PAGE, 19C shows the results of cleavage with a single protease, and 19D shows the results of cleavage with dual proteases. "M" on the figures represents a protein standard marker. On Figure 19C, "1" represents the protein before cleavage with a protease, "2" represents the protein after cleavage with uPA, "3" represents the protein after cleavage with MMP-2, and "4" represents the protein after cleavage with matriptase. On Figure 19D, "1" represents the protein before cleavage with a protease, "2" represents the protein after cleavage with uPA, "3" represents the protein after cleavage with MMP-2, and "4" represents the protein after dual cleavage with uPA and MMP-2. [Figure 19-2] Same as above. [Figure 19-3] Same as above. [Figure 20-1] 20A-20D illustrate representative HPLC analysis of the purified protein. [Figure 20-2] 20A-20D illustrate representative HPLC analysis of the purified protein. [Figure 21-1] Figures 21A-21Q illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 21-2]Figures 21A-21Q illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 21-3] Figures 21A-21Q illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 21-4] Figures 21A-21Q illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 22] Figures 22A-22C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 22A shows the results of non-reducing SDS-PAGE, 22B shows the results of reducing SDS-PAGE, and 22C shows the results of cleavage. "M" on the figure represents a protein standard marker. On Figure 22C, "1" represents the protein before cleavage by TEV, and "2" represents the protein after cleavage by TEV. [Figure 23-1] 23A-23D illustrate representative HPLC analysis of the purified protein. [Figure 23-2] 23A-23D illustrate representative HPLC analysis of the purified protein. [Figure 24] 24A-24D illustrate the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Diagram 25]Figures 25A-25C show SDS-PAGE results for purified proteins and for cleavage of IL-2 fusion protein. 25A shows the results of non-reducing SDS-PAGE, 25B shows the results of reducing SDS-PAGE, and 25C shows the results of cleavage. "M" on the figures represents a protein standard marker. On Figure 25C, "1" represents the protein before cleavage with protease, "2" represents the protein after cleavage with MMP-2, "3" represents the protein after cleavage with uPA, and "4" represents the protein after cleavage with matriptase. [Figure 26-1] 26A-26D illustrate representative HPLC analysis of the purified protein. [Figure 26-2] 26A-26D illustrate representative HPLC analysis of the purified protein. [Figure 27] Figures 27A-27D show the results of SDS-PAGE and HPLC. The band indicates the results of non-reducing SDS-PAGE, the band indicates the results of reducing SDS-PAGE, the band indicates the results of cleavage, and the band indicates the results of HPLC analysis. "M" on the figures represents a protein standard marker. On Figure 27C, "1" represents the protein before cleavage by TEV, and "2" represents the protein after cleavage by TEV. [Figure 28] FIG. 28 illustrates the activity of IL-2 fusion proteins on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 29] 29A-29B show the results of SDS-PAGE for the purified protein. 29A shows the results of non-reducing SDS-PAGE, and 29B shows the results of reducing SDS-PAGE. "M" on the figure represents a protein standard marker. [Diagram 30] Figure 30 shows the results of cleavage by MMP-2. "M" on the figure represents a protein standard marker. "1" represents the protein before cleavage by MMP-2, and "2" represents the protein after cleavage by MMP-2. [Figure 31-1]Figures 31A-31J illustrate representative HPLC analysis of the purified proteins. [Figure 31-2] Figures 31A-31J illustrate representative HPLC analysis of the purified proteins. [Figure 31-3] Figures 31A-31J illustrate representative HPLC analysis of the purified proteins. [Figure 31-4] Figures 31A-31J illustrate representative HPLC analysis of the purified proteins. [Figure 31-5] Figures 31A-31J illustrate representative HPLC analysis of the purified proteins. [Figure 32-1] 32A-32M illustrate the activity of IL-2 proprotein on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 32-2] 32A-32M illustrate the activity of IL-2 proprotein on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). [Figure 32-3] 32A-32M illustrate the activity of IL-2 proprotein on M-07e proliferation as determined by a colorimetric assay (Cell Counting Kit-8 (CCK-8)). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] Detailed Description Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Any methods, materials, compositions, reagents, cells similar or equivalent to those described herein can be used in the practice or testing of the subject matter of this disclosure, but preferred methods and materials are described. All publications and references cited herein, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety as if each individual publication or reference was specifically and individually indicated to be incorporated herein by reference as if fully set forth. Any patent application to which this application claims priority is also incorporated herein by reference in its entirety as described above for publications and references.

[0035] Standard techniques (e.g., electroporation, lipofection) may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation. Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. These and related techniques and procedures may generally be performed according to conventional methods known in the art and as described in various general and more specific references cited and discussed throughout this specification. Unless specific definitions are provided, the nomenclature utilized in connection with and the experimental procedures and techniques of molecular biology, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are well known and commonly used in the art. Standard techniques may be used for recombinant technology, molecular biology, microbiology, chemical synthesis, chemical analysis, pharmaceutical preparation, formulation, and delivery, and patient treatment.

[0036] For purposes of this disclosure, the following terms are defined below.

[0037] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, "a element" includes "one element," "one or more elements," and / or "at least one element."

[0038] "About" means an amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by about 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference amount, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0039] The terms "activatable proprotein", "activatable prodrug", "prodrug", or "proprotein" are used interchangeably herein and refer to an activatable proprotein comprising at least a masking moiety and an active domain, or a derivative / variant thereof, as described herein. In one embodiment, the proprotein may also comprise one or more protein domains.

[0040] The term "antigen" refers to a molecule or a portion of a molecule that can be bound by a selective binding agent, such as an antibody, and can be used in an animal to produce an antibody that can further bind to the epitope of the antigen. An antigen can have one or more epitopes. As used herein, the term "antigen" includes a substance that can induce an immune response against the substance under appropriate conditions and react with the product of the immune response. More broadly, the term "antigen" includes any substance to which an antibody binds or for which an antibody is desired, regardless of whether the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods independent of any immune response.

[0041] "Antagonist" refers to a biological structure or chemical agent that interferes with or otherwise reduces the physiological action of another agent or molecule. In some instances, an antagonist specifically binds to the other agent or molecule. Full and partial antagonists are included.

[0042] "Agonist" refers to a biological structure or chemical agent that increases or enhances the physiological effect of another agent or molecule. In some instances, agonists specifically bind to other agents or molecules. Full and partial agonists are included.

[0043] As used herein, the term "amino acid" is intended to mean both naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimetics. Naturally occurring amino acids include the 20 (L)-amino acids utilized in protein biosynthesis, as well as other amino acids, such as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, homocysteine, citrulline and ornithine. Non-naturally occurring amino acids include, for example, (D)-amino acids, norleucine, norvaline, p-fluorophenylalanine, ethionine, etc., which are known to those skilled in the art. Amino acid analogs include modified forms of naturally occurring and non-naturally occurring amino acids. Such modifications may include, for example, substitution or replacement of chemical groups and chemical moieties on the amino acid, or derivatization of the amino acid. Amino acid mimetics include organic structures that exhibit functionally similar properties, such as, for example, charge and charge spacing characteristics of the reference amino acid. For example, an organic structure that mimics arginine (Arg or R) will have a positively charged portion that is located at a similar molecular distance and has the same degree of flexibility as the e-amino group of the side chain of the naturally occurring Arg amino acid. Mimetics also include structures that are constrained to maintain optimal spacing and charge interactions of amino acids or amino acid functional groups. Those skilled in the art will know or be able to determine which structures constitute functionally equivalent amino acid analogs and amino acid mimetics.

[0044] As used herein, a subject that is "at risk" of developing a disease or adverse reaction may or may not have detectable disease or symptoms of a disease, and may or may not show detectable disease or symptoms of a disease before the treatment method described herein. "At risk" indicates that a subject has one or more risk factors that are measurable parameters that correlate with the occurrence of a disease, as described herein and known in the art. A subject that has one or more of these risk factors has a higher probability of developing a disease or adverse reaction than a subject that does not have one or more of these risk factors.

[0045] "Biocompatible" refers to a material or compound that is not generally deleterious to the biological functions of a cell or subject and does not cause any degree of unacceptable toxicity, including allergic and disease conditions.

[0046] The term "binding" refers to a direct association between two molecules, for example, by covalent, electrostatic, hydrophobic, and ionic and / or hydrogen bonding interactions, including interactions such as salt bridges and water bridges.

[0047] "Coding sequence" means any nucleic acid sequence that contributes to the coding of the polypeptide product of a gene. In contrast, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly contribute to the coding of the polypeptide product of a gene.

[0048] Throughout this disclosure, unless the context requires otherwise, the terms "comprise", "comprises", and "comprising" are understood to mean the inclusion of a stated step or element or group of steps or elements, but not to the exclusion of any other step or element or group of steps or elements.

[0049] "Consisting of" means including and limited to whatever follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the recited elements are required or essential, and that no other elements may be present. "Consisting essentially of" means including any elements recited after this phrase, and limited to other elements that do not interfere with or contribute to an activity or action not specified in the disclosure of the recited elements. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optionally present and may or may not be present depending on whether they substantially affect the activity or action of the recited elements.

[0050] The term "endotoxin-free" or "substantially endotoxin-free" generally refers to compositions, solvents, and / or containers that contain at most trace amounts of endotoxin (e.g., amounts that have no clinically adverse physiological effects on a subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically gram-negative bacteria, although endotoxins can also be found in gram-positive bacteria, such as Listeria monocytogenes. The most widely recognized endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS), which are found in the outer membrane of various gram-negative bacteria and represent a central pathogenic feature in the ability of these bacteria to cause disease. Small amounts of endotoxin in humans cause fever, a drop in blood pressure, and activation of inflammation and coagulation, among other adverse physiological effects.

[0051] Therefore, in pharmaceutical production, it is often desirable to remove most or all traces of endotoxin from drug products and / or drug containers, since even very small amounts can cause adverse effects in humans. Depyrogenation ovens can be used for this purpose, since temperatures above 300°C are typically required to break down most endotoxins. For example, based on primary packaging materials such as syringes or vials, a combination of a glass temperature of 250°C and a holding time of 30 minutes is often sufficient to achieve a 3-log reduction in endotoxin levels. Other methods of removing endotoxin are contemplated, including, for example, chromatography and filtration methods described herein and known in the art.

[0052] Endotoxin can be detected using routine techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for detecting the presence of endotoxin. In this test, very low levels of LPS can cause detectable coagulation of the horseshoe crab extract through a powerful enzyme cascade that amplifies this reaction. Endotoxin can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be less than about 0.001 EU, less than about 0.005 EU, less than about 0.01 EU, less than about 0.02 EU, less than about 0.03 EU, less than about 0.04 EU, less than about 0.05 EU, less than about 0.06 EU, less than about 0.08 EU, less than about 0.09 EU, less than about 0.1 EU, less than about 0.5 EU, less than about 1.0 EU, less than about 1.5 EU, less than about 2 EU, less than about 2.5 EU, less than about 3 EU, less than about 4 EU, less than about 5 EU, less than about 6 EU, less than about 7 EU, less than about 8 EU, less than about 9 EU, or less than about 10 EU per mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1 to 10 EU.

[0053] The term "half maximal effective concentration" or "EC 50" refers to the concentration of an agent (e.g., an activatable proprotein) described herein that induces a response halfway between baseline and maximum after a specified exposure time; thus, the EC 50 represents the concentration of a compound at which 50% of its maximal effect is observed. EC 50 The EC also represents the plasma concentration required to obtain 50% of the maximum effect in vivo. 90 "EC" refers to the concentration of an agent or composition at which 90% of its maximum effect is observed. 90 " is "E.C. 50 " and the Hill slope, or can be determined directly from the data using routine knowledge in the art. In some embodiments, the EC 50 is less than about 0.01 nM, less than about 0.05 nM, less than about 0.1 nM, less than about 0.2 nM, less than about 0.3 nM, less than about 0.4 nM, less than about 0.5 nM, less than about 0.6 nM, less than about 0.7 nM, less than about 0.8 nM, less than about 0.9 nM, less than about 1 nM, less than about 2 nM, less than about 3 nM, less than about 4 nM, less than about 5 nM, less than about 6 nM, less than about 7 nM, less than about 8 nM, less than about 9 nM, less than about 10 nM, In some embodiments, the agent has an EC of about 1 nM or less. 50 It has a value.

[0054] "Immune response" refers to any immunological response originating from the immune system, including responses from cellular and humoral, innate and adaptive immune systems. Exemplary cellular immune cells include, for example, lymphocytes, macrophages, T cells, B cells, NK cells, neutrophils, eosinophils, dendritic cells, mast cells, monocytes, and all subsets thereof. Cellular responses include, for example, effector functions, cytokine release, phagocytosis, efferocytosis, translocation, trafficking, proliferation, differentiation, activation, suppression, cell-cell interactions, apoptosis, and the like. Humoral responses include, for example, IgG, IgM, IgA, IgE responses and their corresponding effector functions.

[0055] The "half-life" of an agent, such as an activatable proprotein, can refer to the time it takes for the agent to lose half of its pharmacological, physiological, or other activity, compared to such activity upon administration to the serum or tissues of an organism, or compared to any other defined time point. "Half-life" can also refer to the time it takes for the amount or concentration of the agent to be half of the starting amount administered to the serum or tissues of an organism, compared to such amount or concentration upon administration to the serum or tissues of an organism, or compared to any other defined time point. Half-life can be measured in serum and / or in any one or more selected tissues.

[0056] The terms "modulate" and "alter" typically include "increasing," "enhancing," or "stimulating," as well as "decreasing" or "reducing" in a statistically significant or physiologically significant amount or degree compared to a control. An "increased," "stimulated," or "enhanced" amount is typically a "statistically significant" amount and can include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more (e.g., 500-fold, 1000-fold) (including all integers and ranges therebetween, e.g., 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, etc.) of the amount produced without the composition (e.g., in the absence of an agent) or by a control composition. A "decreased" or "reduced" amount is typically a "statistically significant" amount and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease (including all integers and ranges therebetween) of the amount produced without the composition (e.g., in the absence of an agent) or by a control composition. Examples of comparisons and "statistically significant" amounts are described herein.

[0057] The terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to a polymer of amino acids, not limited to any particular length. The term "enzyme" includes polypeptide or protein catalysts. These terms include modifications such as myristoylation, sulfation, glycosylation, phosphorylation, and addition or deletion of signal sequences. The term "polypeptide" or "protein" refers to one or more chains of amino acids, each chain comprising amino acids covalently linked by peptide bonds, said polypeptide or protein may comprise multiple chains linked together non-covalently and / or covalently by peptide bonds, having the sequence of a native protein, i.e. a protein occurring in nature and specifically produced by non-recombinant cells, or a protein produced by genetic engineering or recombinant cells, and may include molecules having the amino acid sequence of a native protein, or molecules having deletions, additions, and / or substitutions of one or more amino acids of the native sequence. In certain embodiments, a polypeptide is a "recombinant" polypeptide produced by a recombinant cell that contains one or more recombinant DNA molecules that are typically composed of a heterologous polynucleotide sequence or combination of polynucleotide sequences not otherwise found within the cell.

[0058] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. The terms typically refer to polymeric forms of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides, or modified forms of either type of nucleotide. The terms include single-stranded and double-stranded forms of DNA. The terms "isolated DNA" and "isolated polynucleotide" and "isolated nucleic acid" refer to molecules that are isolated free of total genomic DNA of a particular species. Thus, an isolated DNA segment that encodes a polypeptide refers to a DNA segment that contains one or more coding sequences, yet is substantially isolated or purified from the total genomic DNA of the species from which the DNA segment is obtained. Similarly, non-coding polynucleotides that do not encode a polypeptide (e.g., primers, probes, oligonucleotides) are also included. Also included are recombinant vectors, including, for example, expression vectors, viral vectors, plasmids, cosmids, phagemids, phages, viruses, etc.

[0059] Additional coding or non-coding sequences may, but need not, be present within the polynucleotides described herein, and the polynucleotides may, but need not, be linked to other molecules and / or supporting materials. Thus, a polynucleotide or expressible polynucleotide, regardless of the length of the coding sequence itself, may be combined with other sequences, such as expression control sequences.

[0060] The term "isolated" polypeptide or protein as referred to herein means that the subject protein is (1) free of at least some other proteins with which it is typically found in nature, (2) essentially free of other proteins from the same source, e.g., from the same species, (3) expressed by cells from a different species, (4) separated from at least about 50% of the polynucleotides, lipids, carbohydrates, or other materials with which it is naturally associated, (5) not associated (by covalent or non-covalent interactions) with portions of proteins with which it is naturally associated, (6) operably associated (by covalent or non-covalent interactions) with which it is not naturally associated, or (7) not naturally occurring. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, or may be of synthetic origin, or any combination thereof. In certain embodiments, an isolated protein is substantially free of proteins or polypeptides or other contaminants found in its natural environment that would interfere with its use (therapeutic, diagnostic, prophylactic, research, or otherwise).

[0061] In certain embodiments, the "purity" of any given agent (e.g., an activatable proprotein) in a composition may be defined. For example, a certain composition may contain an agent, such as a polypeptide agent, that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure on a protein basis or weight-by-weight basis, including all decimal points and ranges therebetween, as measured, for example, but in no way limited to, by high performance liquid chromatography (HPLC), a well-known form of column chromatography often used to separate, identify, and quantify compounds in biochemistry and analytical chemistry.

[0062] The term "reference sequence" generally refers to a nucleic acid coding sequence or amino acid sequence to which another sequence is compared. All polypeptide and polynucleotide sequences described herein, including those sequences described by name and those sequences described in the tables and sequence listing, are included as reference sequences.

[0063] Certain embodiments include biologically active "variants" and "fragments" of the proteins / polypeptides and polynucleotides encoding them described herein. "Variants" contain one or more substitutions, additions, deletions, and / or insertions compared to a reference polypeptide or polynucleotide (see, e.g., Tables and Sequence Listings). A variant polypeptide or polynucleotide comprises an amino acid or nucleotide sequence having at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more sequence identity or similarity or homology to a reference sequence described herein and substantially retains the activity of the reference sequence. Also included are sequences that consist of a reference sequence or that differ from the reference sequence by the addition, deletion, insertion, or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more amino acids or nucleotides, and that substantially retain at least one activity of the reference sequence. In certain embodiments, the addition or deletion comprises a C-terminal and / or N-terminal addition and / or deletion.

[0064] The term "sequence identity", or including, for example, "50% identical sequence", as used herein, refers to the degree to which sequences are identical on a nucleotide-by-nucleotide basis or on an amino acid-by-amino acid basis over a comparison window.Thus, "sequence identity percentage" can be calculated by: comparing two optimally aligned sequences over a comparison window; determining the number of positions where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to obtain the number of matched positions; dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size); and multiplying the result by 100 to obtain the percentage of sequence identity. Optimal alignment of sequences for aligning a comparison window can be performed by computerized algorithm implementation (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA), or by inspection and best alignment (i.e., resulting in the highest percentage of homology for the comparison window) generated by any of the various methods selected. For example, BLAST family programs disclosed in Altschul et al., Nucl. Acids Res. 25:3389, 1997 may also be referenced.

[0065] The term "solubility" refers to the property of an agent (e.g., an activatable proprotein) provided herein to dissolve in a liquid solvent to form a homogenous solution. Solubility is typically expressed as a concentration, either by mass of solute per unit volume of solvent (g solute / kg solvent, g / dL (100 mL), mg / ml, etc.), molality, molality, mole fraction, or other similar concentration description. The maximum equilibrium amount of solute that can be dissolved per volume of solvent is the solubility of that solute in that solvent under specified conditions including temperature, pressure, pH, and the nature of the solvent. In certain embodiments, solubility is measured at physiological pH, or other pHs, such as pH 5.0, pH 6.0, pH 7.0, pH 7.4, pH 7.6, pH 7.8, or pH 8.0 (e.g., about pH 5-8). In certain embodiments, solubility is measured in water or PBS or NaCl (NaPO 4 In a specific embodiment, the solubility is measured in a physiological buffer, such as a relatively low pH (e.g., pH 6.0) and a relatively high salt (e.g., 500 mM NaCl and 10 mM NaPO 4) in a biological fluid (solvent) such as blood or serum. In certain embodiments, the temperature can be about room temperature (e.g., about 20° C., about 21° C., about 22° C., about 23° C., about 24° C., about 25° C.) or about body temperature (37° C.). In certain embodiments, the agent has a solubility of at least about 0.1 mg / ml, at least about 0.2 mg / ml, at least about 0.3 mg / ml, at least about 0.4 mg / ml, at least about 0.5 mg / ml, at least about 0.6 mg / ml, at least about 0.7 mg / ml, at least about 0.8 mg / ml, at least about 0.9 mg / ml, at least about 1 mg / ml, at least about 2 mg / ml, at least about 3 mg / ml, at least about 4 mg / ml, at least about 5 mg / ml, at least about 6 mg / ml, at least about 7 mg / ml, at least about 8 mg / ml, at least about ...1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at least about 1 mg / ml, at In some embodiments, the solubility is 0 mg / ml, at least about 11 mg / ml, at least about 12 mg / ml, at least about 13 mg / ml, at least about 14 mg / ml, at least about 15 mg / ml, at least about 16 mg / ml, at least about 17 mg / ml, at least about 18 mg / ml, at least about 19 mg / ml, at least about 20 mg / ml, at least about 25 mg / ml, at least about 30 mg / ml, at least about 40 mg / ml, at least about 50 mg / ml, at least about 60 mg / ml, at least about 70 mg / ml, at least about 80 mg / ml, at least about 90 mg / ml, or at least about 100 mg / ml.

[0066] A "subject" or "subject in need thereof" or "patient" or "patient in need thereof" includes a mammalian subject, such as a human subject.

[0067] "Substantially" or "essentially" means nearly entirely or completely, e.g., 95%, 96%, 97%, 98%, 99%, or more of some given amount.

[0068] "Statistically significant" means that the result is unlikely to have occurred by chance. Statistical significance can be determined by any method known in the art. Commonly used measures of significance include p-value, which is the frequency or probability that an observed event occurs if the null hypothesis is true. If the p-value obtained is less than the significance level, the null hypothesis is rejected. In a simple example, the significance level is defined as a p-value of 0.05 or less.

[0069] "Therapeutic response" refers to an improvement in symptoms (whether sustained or not) upon administration of one or more therapeutic agents.

[0070] As used herein, the terms "therapeutically effective amount," "therapeutic dose," "prophylactically effective amount," or "diagnostically effective amount" are the amount of an agent (e.g., an activatable proprotein, an activator protein) required to elicit a desired biological response following administration.

[0071] As used herein, "treatment" of a subject (e.g., a mammal, e.g., a human) or cell is any type of intervention used in an attempt to change the natural course of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition, and may be performed prophylactically or after the initiation of a pathological event or after contact with a pathogenic agent. Also included is "prophylactic" treatment, which may be directed to reducing the rate of progression of the disease or condition being treated, delaying the onset of the disease or condition, or reducing the severity of its onset. "Treatment" or "prevention" does not necessarily indicate a complete eradication, cure, or prevention of the disease or condition, or its associated symptoms.

[0072] The term "wild type" refers to a gene or gene product (eg, a polypeptide) that is most frequently observed in a population, and is thus arbitrarily designed the "normal" or "wild type" form of the gene.

[0073] Each embodiment herein applies to every other embodiment unless expressly stated otherwise.

[0074] Activatable Proproteins The embodiments of the present disclosure relate to activatable proprotein homodimers or prodrugs comprising two IL-2 proteins that remain relatively inactive in the proprotein form and can be activated upon contact with an appropriate environment. The activatable proproteins described herein comprise at least two separate but otherwise identical (or substantially identical) polypeptide chains that are bound to each other through non-covalent interactions and / or certain covalent bonds, e.g., disulfide bonds, but not through peptide or amide bonds. Generally, each polypeptide chain comprises an IL-2 protein, an IL-2 binding protein, e.g., an IL-2Rα protein, and a cleavable linker. Here, the IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and the IL-2 protein of the second polypeptide binds to the IL-2 binding protein of the first polypeptide to form a relatively stable homodimer in which these binding interactions sterically hinder the interaction or binding of the IL-2 protein in each chain with its cognate receptor on a cell (see, e.g., Figures 2A and 2C). In some examples, each polypeptide chain comprises a purification tag at the N- or C-terminus, which is separated from the remainder of the polypeptide by a linker (see, e.g., Figures 2B and 3B). In some examples, each polypeptide chain comprises a binding domain (e.g., an Fc domain or fragment thereof) at the N- or C-terminus, which is separated from the remainder of the polypeptide by a linker (see, e.g., Figures 5A-5D), which binds to a binding domain on the other polypeptide chain to further stabilize the proprotein homodimer (see, e.g., Figures 2C, 2E, 3C, and 3D). As described above, at least one of the linkers is a cleavable linker that, when cleaved in the target cell or tissue, restores IL-2 activity by opening the homodimer to expose at least one active or binding site of the IL-2 protein.The IL-2 portion of the now activated protein(s) can thereby interact or bind with a portion of its cognate receptor(s), e.g., the IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains on an immune cell, thereby affecting downstream immune cell signaling pathways.

[0075] The activatable proproteins described herein are capable of inhibiting high initial serum C levels that cause overactivation of the immune system. max , addresses many of the shortcomings of standard IL-2 therapy in the treatment of cancer, infectious diseases and other diseases, including preferential activation of regulatory T cells expressing the IL-2Rα / β / γc receptor chain compared to immune cells expressing the IL-2Rβ / γc receptor chain, poor PK due to the small molecular size of IL-2 and / or catabolism of the large number of immune cells expressing IL-2 receptors, poor accumulation in target tissues (e.g., cancer, tumors) due to poor PK and / or ineffective tumor targeting, and undesirable accumulation and immune activation in normal tissues.

[0076] Thus, an embodiment of the present disclosure is an activatable proprotein homodimer (complex) comprising a first polypeptide (chain) and a second polypeptide (chain), the first polypeptide and the second polypeptide comprise, in N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein; or the first polypeptide and the second polypeptide comprise, in N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein; The present invention relates to an activated proprotein homodimer comprising an IL-2 protein that is capable of binding to an IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell in vitro or in vivo, the IL-2 protein of the first polypeptide being capable of binding to an IL-2Rβ / γc and / or IL-2Rα / β / γc chain present on the surface of an immune cell ...

[0077] An activatable proprotein homodimer (complex) comprising a first polypeptide (chain) and a second polypeptide (chain), the first and second polypeptides comprise, in N-to-C-terminal or C-to-N-terminal direction, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and optionally an affinity purification tag; or the first and second polypeptides comprise, in N-to-C-terminal or C-to-N-terminal direction, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and optionally an affinity purification tag; The IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and the IL-2 binding protein of the first polypeptide binds to the IL-2 protein of the second polypeptide, and said (total) binding is in vitro or in vivo. Also included are activatable proprotein homodimers which mask the binding site of an IL-2 protein(s) that would otherwise bind to the IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vivo, and in which the first linker is a cleavable linker.

[0078] As described above, the IL-2 protein(s) and the IL-2 binding protein(s) interact or bind to one another, for example, via non-covalent interactions or certain covalent bonds (e.g., disulfide bonds). In some examples, the binding of the IL-2 protein(s) to the IL-2 binding protein(s), for example, the IL-2Rα protein(s), activates regulatory T cells (T reg ) to its cognate IL-2Rα / β / γc receptor chains expressed on the T reg This can provide the advantage of minimizing activation of proproteins and reducing consumption of proproteins and similar active proteins. Exemplary IL-2 proteins and IL-2 binding proteins are described elsewhere herein.

[0079] In some examples, the binding moieties of the first and second polypeptides form a dimer together through at least one non-covalent interaction, at least one covalent bond (e.g., at least one disulfide bond), or any combination of non-covalent interactions and covalent bonds to further stabilize the activatable proprotein and / or further mask the binding of the IL-2 protein to its cognate receptor, e.g., IL-2Rα / β / γc and / or IL-2Rβ / γc receptor chain. Typically, however, the binding moieties of the first and second polypeptides do not bind to each other through peptide or amide bonds, nor do they form a dimer. In some embodiments, the binding moieties bind to each other as heterodimers, i.e., as a heterodimer composed of two different binding moieties. In some embodiments, the binding moieties bind to each other as homodimers, i.e., as a homodimer composed of two identical or nearly identical binding moieties. Thus, the binding moieties of the first and second polypeptides can be the same (or substantially the same) or different. In most instances, the binding moieties of the first and second polypeptides are the same and do not bind to either the IL-2 protein or the IL-2 binding protein. However, in some instances, one or both of the binding moieties can bind to the IL-2 protein and / or the IL-2 binding protein. Exemplary binding moiety structures are described herein.

[0080] As described above, at least one of the linkers comprises a cleavable linker, e.g., a linker that can be cleaved by a protease. In some examples, one linker comprises a cleavable linker and the other linker is a stable (e.g., physiologically stable) linker. In some examples, both linkers comprise a cleavable linker. In some examples, the protease is expressed in a target tissue or cell, e.g., a cancer tissue or cell. Cleavage of the linker in that situation releases the masking moiety, removing steric hindrance of the IL-2 protein, allowing selective activation of the IL-2 protein in diseased tissue or cell compared to normal or healthy tissue or cell. Such selective and localized activation not only increases the half-life of the administered IL-2 by reducing unnecessary consumption thereof, but also enhances the tissue penetration of IL-2 and reduces undesirable systemic effects, among other advantages. Exemplary linkers are described herein.

[0081] In some embodiments, the homodimeric binding between the first and second polypeptides allosterically inhibits binding of the IL-2 protein to its target, e.g., the cognate IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains on the surface of an immune cell. In these and related embodiments, the activatable proprotein exhibits no or substantially no binding to its target, or exhibits 0.001% or less, 0.01% or less, 0.1% or less, 1% or less, 2% or less, 3% or less, 4% or less, 5% or less, 6% or less, 7% or less, 8% or less, 9% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, or 50% or less binding compared to the binding of the active domain or IL-2 protein alone, optionally in vivo or using Target Displacement in vivo techniques available in the art. or at least 2 hours, at least 4 hours, at least 6 hours, at least 8 hours, at least 12 hours, at least 28 hours, at least 24 hours, at least 30 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, or at least 5 days, at least 10 days, at least 15 days, at least 30 days, at least 45 days, at least 60 days, at least 90 days, at least 120 days, at least 150 days, at least 180 days, or at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, or more, as appropriate, when measured by an in vitro assay.

[0082] The various components of each polypeptide chain can be fused in any direction. For example, in some embodiments, the first and second polypeptides comprise, from N to C terminal, a binding moiety, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein. In some embodiments, the first and second polypeptides comprise, from N to C terminal, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and a binding moiety. In certain embodiments, the first and second polypeptides comprise, from N to C terminal, a binding moiety, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein. In some embodiments, the first and second polypeptides comprise, from N to C terminal, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and a binding moiety. In certain embodiments, the first and second polypeptides comprise, from N to C terminal, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and an affinity purification tag. In some embodiments, the first and second polypeptides of (d) comprise, from N to C terminal, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and an affinity purification tag. Other possible orientations will be apparent to those skilled in the art.

[0083] Certain activatable proproteins are composed of only two of the foreign protein chains, i.e., they are composed of only the first and second polypeptides described herein. However, in some cases, certain activatable proproteins comprise multiple chains, e.g., the first and second polypeptide chains form a "core structure" on which additional or higher order structures can be built, and the various core structures are optionally linked to each other via additional protein binding domains.

[0084] The individual components of the activatable proprotein are described in more detail herein below.

[0085] IL-2 Protein. The activatable proproteins described herein contain at least one "IL-2 protein" (or interleukin-2 protein), including human IL-2 protein. IL-2 is a cytokine that signals through the IL-2 receptor (IL-2R), a complex composed of up to three chains called the α (CD25), β (CD122), and γc (CD132) chains. IL-2 is produced by T cells in response to antigenic or mitogenic stimulation and is required for T cell proliferation and other activities that are crucial for regulating the immune response. IL-2 can stimulate B cells, monocytes, lymphokine-activated killer cells, natural killer cells, and glioma cells, among other immune cells.

[0086] IL-2 is a 15-16 kDA protein composed of a signal peptide (residues 1-20) and an active mature protein (residues 21-153). An exemplary human IL-2 amino acid sequence is provided below in Table S1. [Table S1-1] [Table S1-2] [Table S1-3]

[0087] Thus, in certain embodiments, the IL-2 protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S1, or an active variant or fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a sequence selected from Table S1. In some embodiments, an "active" IL-2 protein or fragment or variant is characterized by its ability to bind to the IL-2Rβ / γc and IL-2Rα / β / γc receptor chains present on the surface of immune cells, e.g., in vitro or in vivo, and its ability to stimulate downstream signaling activity in the absence of steric hindrance by a masking moiety as described herein. Examples of downstream signaling activity include IL-2-mediated signaling through one or more of the JAK-STAT, PI3K / Akt / mTOR, and MAPK / ERK pathways, including combinations thereof. Overall, IL-2 signaling stimulates a series of downstream pathways that lead to responses that have important roles in the development, function, and survival of CD4 T cells, CD8 T cells, NK cells, NKT cells, macrophages, and intestinal intraepithelial lymphocytes, among others.

[0088] In certain embodiments, the IL-2 protein is a mature form of IL-2, or an active variant or fragment thereof, comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 21-153 of SEQ ID NO: 1. In some embodiments, the IL-2 protein comprises a C145X substitution as defined by SEQ ID NO: 1, where X is any amino acid. In specific embodiments, the IL-2 protein comprises a C145S substitution as defined by SEQ ID NO: 1.

[0089] Certain IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:2 (mature human IL-2 with a C125S substitution). In some embodiments, an active variant or fragment of SEQ ID NO:2 retains the S125 residue as defined therein.

[0090] Certain IL-2 proteins contain one or more defined amino acid substitutions compared to the exemplary amino acid sequences in Table S1. For example, some IL-2 proteins contain one or more amino acid substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined by SEQ ID NO:2. In some embodiments, the IL-2 protein forms a disulfide bond with an IL-2 binding protein (e.g., IL-2Ra) through one or more cysteine ​​substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C. Certain IL-2 proteins contain one or more amino acid substitutions at positions 69, 74, and / or 128 as defined by SEQ ID NO:2, including combinations thereof, including when the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO:2. Some IL-2 proteins include one or more amino acid substitutions at positions R38, F42, Y45, E62, E68, and / or L72 as defined by SEQ ID NO:2, including combinations thereof, such as R38A and R38K; F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42G, F42H ... D, F42R, F42K, and F42I; Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K; E62A and E62L; E68A and E68V; and L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K. Specific examples include where the IL-2 protein comprises one or a combination of amino acid substitutions selected from: F42A, Y45A, and L72G; R38K, F42Q, Y45N, E62L, and E68V; R38K, F42Q, Y45E, and E68V; R38A, F42I, Y45N, E62L, and E68V; R38K, F42K, Y45R, E62L, and E68V; R38K, F42I, Y45E, and E68V; and R38A, F42A, Y45A, and E62A.Some IL-2 proteins include one or a combination of amino acid substitutions at T3 and / or E61 as defined by SEQ ID NO: 2, e.g., T3A and / or E61S. Thus, an IL-2 protein can include any one or more of the foregoing amino acid substitutions, including combinations thereof.

[0091] It will be appreciated that any one or more of the aforementioned IL-2 proteins can be combined with other components described herein, such as IL-2 binding proteins, e.g., IL-2Rα proteins, masking moieties including binding moieties and linkers, and any other optional protein domains, to generate one or more activatable proproteins or larger multi-chain structures comprising the same.

[0092] IL-2 binding proteins. The activatable proproteins described herein include at least one "IL-2 binding protein." Examples of IL-2 binding proteins include IL-2Rα proteins, including human IL-2Rα proteins, and antibodies and antigen-binding fragments thereof that bind to the IL-2 proteins described herein.

[0093] In certain embodiments, the IL-2 binding protein is a human IL-2Rα protein, or a variant or fragment thereof that binds to the IL-2 protein. Exemplary human IL-2Rα amino acid sequences are provided in Table S2 below. [Table S2-1] [Table S2-2]

[0094] Thus, in certain embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence selected from Table S2, or an active variant or fragment thereof that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a sequence selected from Table S2 and that binds to IL-2 protein. In some embodiments, the IL-2Rα protein comprises, consists of, or consists essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 22-187 or 22-240 of SEQ ID NO:4 (full-length wild-type human IL-2Rα).

[0095] Certain IL-2Rα proteins contain one or more defined amino acid substitutions compared to the exemplary amino acid sequences in Table S2. For example, in some examples, the IL-2Rα protein contains one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C as defined by SEQ ID NO:6 (human IL-2Rα Sushi1-Sushi2 domains). In some examples, the IL-2Rα protein contains an alanine substitution at position 49 and / or 68 as defined by SEQ ID NO:6. In some embodiments, the IL-2Rα protein contains a K38S substitution as defined by SEQ ID NO:6. Thus, the IL-2Rα protein can contain any one or more of the aforementioned amino acid substitutions, including combinations thereof.

[0096] In certain of these and related embodiments, the IL-2Rα protein forms at least one disulfide bond with the IL-2 protein via one or more of the aforementioned cysteines and one or more cysteines in the IL-2 protein. In specific embodiments, the IL-2Rα and IL-2 proteins form disulfides, at least one disulfide bond, between one or more pairs of cysteines selected from IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, IL2-R38C and IL2Rα-H120C, IL2-T41C and IL2Rα-I118C, IL2-F42C and IL2Rα-N27C, IL2-E61C and IL2Rα-K38C, IL2-E61C and IL2Rα-S39C, and IL2-V69C and IL2Rα-L42C. In certain embodiments, as described above, the bond (e.g., a disulfide bond) between the IL-2 protein and the IL-2Rα protein is a T reg The masking moiety masks or sterically hinders the binding site of the IL-2 protein that preferentially binds to the IL-2Rα / β / γc chain expressed on the T. In some instances, the active or activated form of the protein retains the binding between the IL-2 protein and the IL-2Rα protein after cleavage of at least one linker and release of the corresponding masking moiety, thus inhibiting T cell proliferation and differentiation. reg It does not preferentially bind to the IL-2Rα / β / γc chains expressed on

[0097] As noted above, in certain embodiments, the IL-2 binding protein comprises an antibody or an antigen-binding fragment thereof that specifically binds to the IL-2 protein. Examples include whole antibodies, Fab, Fab', F(ab')2, monospecific Fab2, bispecific Fab2, FV, single chain Fv (scFv), scFV-Fc, nanobodies, diabodies, camelid antibodies, and minibodies. In a specific embodiment, the antibody is NARA1 or an antigen-binding fragment thereof (see, e.g., Arenas-Ramirez et al., J. Immunol. 1999, 144:131-132, which are incorporated herein by reference). al., Science Translational Medicine. 8: 367ral66, 2016; and U.S. Patent Application Publication No. 2019 / 0016797). In certain embodiments, and similar to those described above, the bond (e.g., a disulfide bond) between the IL-2 protein and the anti-IL-2 antibody (or antigen-binding fragment thereof) is a T reg The masking moiety masks or sterically hinders the binding site of the IL-2 protein that preferentially binds to the IL-2Rα / β / γc chain expressed on the T. In some instances, the active or activated form of the protein retains the binding between the IL-2 protein and the IL-2Rα protein after cleavage of at least one linker and release of the corresponding masking moiety, thus inhibiting T cell proliferation and differentiation. reg It does not preferentially bind to the IL-2Rα / β / γc chains expressed on

[0098] As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also fragments thereof (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chain (ScFv), synthetic variants thereof, naturally occurring variants, fusion proteins comprising an antibody portion having an antigen-binding fragment of the required specificity, humanized antibodies, chimeric antibodies, and any other modified configuration of an immunoglobulin molecule that contains an antigen-binding site or fragment (epitope recognition site) with the required specificity. Certain features and characteristics of antibodies (and antigen-binding fragments thereof) are described in more detail herein.

[0099] The antibody or antigen-binding fragment can be of essentially any type. As is well known in the art, an antibody is an immunoglobulin molecule that can specifically bind to a target, such as an immune checkpoint molecule, through at least one epitope recognition site located in the variable region of the immunoglobulin molecule.

[0100] The term "antigen-binding fragment" as used herein refers to a polypeptide fragment that contains at least one CDR of an immunoglobulin heavy and / or light chain that binds to an antigen of interest. In this regard, an antigen-binding fragment of an antibody described herein is a V-type polypeptide fragment from an antibody that binds to a target molecule. H and V L It may include one, two, three, four, five, or all six CDRs of the sequence.

[0101] The binding properties of antibodies and antigen-binding fragments thereof can be quantified using methods well known in the art (see Davies et al., Annual Rev. Biochem. 59:439-473, 1990). In some embodiments, the antibody or antigen-binding fragment thereof binds to a target molecule, e.g., an IL-2 protein or an epitope or complex thereof, at about or about 10 -7 M or less ~ about 10 -8 In some embodiments, the equilibrium dissociation constant is in the range of about or about 10 -9 M or less ~ about 10 -10In certain exemplary embodiments, the antibody or antigen-binding fragment thereof has a mAb of about, at least about 0.01 nM or less, about, at least about 0.05 nM or less, about, at least about 0.1 nM or less, about, at least about 0.2 nM or less, about, at least about 0.3 nM or less, about, at least about 0.4 nM or less, about, at least about 0.5 nM or less, about, at least about 0.6 nM or less, about, at least about 0.7 nM or less, about, at least about 0.8 nM or less. M or less, about, at least about 0.9 nM or less, about, at least about 1 nM or less, about, at least about 2 nM or less, about, at least about 3 nM or less, about, at least about 4 nM or less, about, at least about 5 nM or less, about, at least about 6 nM or less, about, at least about 7 nM or less, about, at least about 8 nM or less, about, at least about 9 nM or less, about, at least about 10 nM or less, about, at least about 11 nM or less, about, at least about 12 nM or less, about, at least about 13 nM or less, about, at least about 14 nM or less, about, at least about 15 nM or less, about, at least about 16 nM or less, about, at least about 17 nM or less, about, at least about 18 nM or less, about, at least about 19 nM or less, about, at least about 20 nM or less, about, at least about 21 nM or less, about, at least about 22 nM or less, about, at least or about, at least about 23 nM or less, about, at least about 24 nM or less, about, at least about 25 nM or less, about, at least about 26 nM or less, about, at least about 27 nM or less, about, at least about 28 nM or less, about, at least about 29 nM or less, about, at least about 30 nM or less, about, at least about 40 nM or less, or about, at least about 50 nM or less. 50).

[0102] A molecule, such as a polypeptide or an antibody, is said to exhibit "specific binding" or "preferential binding" if it reacts or associates more frequently, rapidly, for a longer period, and / or with a higher affinity to a particular cell, substance, or particular epitope than it reacts or associates with alternative cells or substances or epitopes. An antibody "specifically binds" or "preferentially binds" to a target molecule or epitope if it binds with higher affinity, avidity, more readily, and / or for a longer period, e.g., in a statistically significant amount, than it binds to other substances or epitopes. Typically, one member of a pair of molecules exhibiting specific binding has an area or depression on its surface that specifically binds to the other member of the pair of molecules and is therefore complementary to its particular spatial and / or polar organization. Thus, the members of the pair have the property of specifically binding to each other. For example, an antibody that specifically or preferentially binds to a particular epitope is an antibody that binds to the particular epitope with higher affinity, avidity, more readily, and / or for a longer period than it binds to other epitopes. Similarly, it will be understood by reading this definition that an antibody (or moiety or epitope) that specifically or preferentially binds, for example, to a first target may or may not specifically or preferentially bind to a second target. The term is also applicable where an antibody is specific for a particular epitope borne by, for example, several antigens, in which case a specific binding member having an antigen-binding fragment or domain may bind to a variety of antigens that bear the epitope, and may be cross-reactive with, for example, several different forms of a target antigen from multiple species that share a common epitope.

[0103] Immunological binding generally refers to the type of non-covalent interaction that occurs between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific, for example and without limitation, as a result of electrostatic, ionic, hydrophilic, and / or hydrophobic attractions or repulsions, steric forces, hydrogen bonds, van der Waals forces, and other interactions. The strength or affinity of an immunological binding interaction can be expressed in terms of the dissociation constant (Kd) of the interaction, with a smaller Kd representing a higher affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, both the "on-rate constant" (Kon) and the "off-rate constant" (Koff) can be determined by calculation of the concentrations and the actual association and dissociation rates. The ratio Koff / Kon allows for the cancellation of all parameters that are not related to affinity, and is thus equal to the dissociation constant Kd. As used herein, the term "affinity" includes the equilibrium constant for the reversible binding of two agents, Kd or EC 50 The affinity of an antibody for an IL-2 protein or epitope can be, for example, from about 100 nanomolar (nM) to about 0.1 nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM to about 1 femtomolar (fM). As used herein, the term "avidity" refers to the resistance of a complex of two or more agents to dissociation upon dilution.

[0104] Antibodies can be prepared by any of a variety of techniques known to those skilled in the art. See, for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988. Monoclonal antibodies specific to a polypeptide of interest can be prepared, for example, using the technique of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and improvements thereto. Also included are methods of expressing human antibodies using transgenic animals such as mice. See, for example, Neuberger et al., Nature Biotechnology 14:826, 1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101, 1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995. A specific example includes the VELOCIMMUNE® platform by REGENEREX® (see, eg, US Pat. No. 6,596,541).

[0105] In certain embodiments, the antibodies and antigen-binding fragments thereof described herein comprise a set of heavy and light chain CDRs interposed between a set of heavy and light chain framework regions (FRs), each of which provides support for the CDRs and defines the spatial relationship of the CDRs relative to each other. As used herein, the term "CDR set" refers to the three hypervariable regions of a heavy or light chain V region. Starting from the N-terminus of the heavy or light chain, these regions are designated as "CDR1", "CDR2", and "CDR3", respectively. Thus, an antigen-binding site comprises six CDRs, comprising a set of CDRs from each of the heavy and light chain V regions. A polypeptide comprising a single CDR (e.g., CDR1, CDR2, or CDR3) is referred to herein as a "molecular recognition unit". Crystal structure analysis of several antigen-antibody complexes has demonstrated that the amino acid residues of the CDRs form extensive contacts with the bound antigen, with the most extensive antigen contact being with the heavy chain CDR3. Thus, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0106] As used herein, the term "FR set" refers to four adjacent amino acid sequences that are the framework for the CDRs of a CDR set of a heavy or light chain V region. Some FR residues may contact the bound antigen. However, FRs, especially those immediately adjacent to the CDRs, are primarily involved in folding the V region into the antigen-binding site. Within FRs, certain amino residues and certain structural features are very highly conserved. In this regard, all V region sequences contain an internal disulfide loop of approximately 90 amino acid residues. When the V region folds into the binding site, the CDRs are displayed as protruding loop motifs that form the antigen-binding surface. It is generally recognized that there are conserved structural regions of FRs that affect the folded shape of the CDR loops into certain "classical" structures, regardless of the exact amino acid sequence of the CDRs. In addition, certain FR residues are known to participate in non-covalent interdomain contacts that stabilize the interaction of antibody heavy and light chains.

[0107] The structure and location of immunoglobulin variable domains can be determined with reference to Kabat, EA et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987, and updates thereof.

[0108] Also included are "monoclonal" antibodies, which refer to a homogeneous antibody population, and which are composed of amino acids (naturally occurring and non-naturally occurring) involved in selective binding of an epitope. Monoclonal antibodies are directed to a single epitope and are highly specific. The term "monoclonal antibody" encompasses not only intact and full-length monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (ScFv), variants thereof, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified configuration of immunoglobulin molecules containing antigen-binding fragments (epitope recognition sites) with the required specificity and ability to bind to the epitope. No limitations are intended as to the origin of the antibody or the manner in which it is made (e.g., hybridoma, phage selection, recombinant expression, transgenic animals). The term includes whole immunoglobulins and fragments such as those listed above under the definition of "antibody".

[0109] The proteolytic enzyme papain preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) contain a covalently linked heterodimer, each of which contains an intact antigen binding site. The enzyme pepsin can cleave IgG molecules to provide several fragments, including the F(ab')2 fragment, which contains both antigen binding sites. Fv fragments for use according to certain embodiments can be produced by preferential proteolytic cleavage of IgM, and more rarely, IgG or IgA immunoglobulin molecules. However, Fv fragments are more commonly derived using recombinant techniques known in the art. Fv fragments contain a non-covalently linked VH::VL heterodimer that contains an antigen binding site that retains much of the antigen recognition and binding capabilities of the native antibody molecule. See Inbar et al., PNAS USA. 69:2659-2662, 1972; Hochman et al., Biochem. 15:2706-2710, 1976; and Ehrlich et al., Biochem. 19:4091-4096, 1980.

[0110] In certain embodiments, single chain Fv (scFV) antibodies are contemplated. For example, kappabodies (Ill et al., Prot. Eng. 10:949-57, 1997); minibodies (Martin et al., EMBO J 13:5305-9, 1994); diabodies (Holliger et al., PNAS 90:6444-8, 1993); or Janusins ​​(Traunecker et al., EMBO J 10:3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992) may be prepared using standard molecular biology techniques following the teachings of the present application for the selection of antibodies with desired specificity.

[0111] Single chain Fv (scFv) polypeptides are covalently linked VH::VL heterodimers expressed from gene fusions containing VH and VL coding genes linked by a peptide-encoding linker. Huston et al. (PNAS USA. 85(16):5879-5883, 1988). Several methods have been described to identify chemical structures for converting naturally aggregated but chemically separated light and heavy polypeptide chains from antibody V regions into scFv molecules that fold into a three-dimensional structure substantially similar to the structure of an antigen-binding site. See, for example, U.S. Patent Nos. 5,091,513 and 5,132,405 to Huston et al.; and U.S. Patent No. 4,946,778 to Ladner et al.

[0112] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein are in the form of "diabodies". Diabodies are multimers of polypeptides, each of which comprises a first domain comprising a binding region of an immunoglobulin light chain and a second domain comprising a binding region of an immunoglobulin heavy chain, and the two domains are linked (e.g., by a peptide linker) but cannot associate with each other to form an antigen-binding site, which is formed by the association of a first domain of one polypeptide in the multimer with a second domain of another polypeptide in the multimer (WO94 / 13804). dAb fragments of antibodies consist of VH domains (Ward et al., Nature 341:544-546, 1989). Diabodies and other multivalent or multispecific fragments can be constructed, for example, by gene fusion (see WO94 / 13804; and Holliger et al., PNAS USA. 90:6444-6448, 1993).

[0113] Also included are minibodies that contain scFv linked to CH3 domains (see Hu et al., Cancer Res. 56:3055-3061, 1996).See also Ward et al., Nature. 341:544-546, 1989; Bird et al., Science. 242:423-426, 1988; Huston et al., PNAS USA. 85:5879-5883, 1988); PCT / US92 / 09965; WO94 / 13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

[0114] When bispecific antibodies are used, they can be conventional bispecific antibodies, which can be produced in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), e.g., prepared chemically or from hybrid hybridomas, or any of the bispecific antibody fragments mentioned above. Diabodies and scFvs can be constructed without Fc regions using only variable domains, potentially reducing the effects of anti-idiotypic reaction.

[0115] Bispecific diabodies may also be particularly useful because, in contrast to bispecific whole antibodies, they can be easily constructed and expressed in E. coli. Diabodies (and many other polypeptides, such as antibody fragments) with appropriate binding specificity can be easily selected from libraries using phage display (WO94 / 13804). For example, if one arm of a diabody specifically directed against antigen X is kept constant, a library can be made in which the other arm is varied, and antibodies of appropriate specificity can be selected. Bispecific whole antibodies can be made by knob-into-hole engineering (Ridgeway et al., Protein Eng., 9:616-621, 1996).

[0116] In certain embodiments, the antibodies or antigen-binding fragments described herein are in the form of UniBody®. UniBody® is an IgG4 antibody with the hinge region removed (GenMab Utrecht, The Netherlands; see, e.g., US20090226421). This antibody technology creates a stable smaller antibody format with a predicted longer therapeutic window than current small antibody formats. IgG4 antibodies are considered inert and do not interact with the immune system. Fully human IgG4 antibodies may be modified by eliminating the hinge region of the antibody, resulting in half-molecule fragments with distinct stability characteristics compared to the corresponding intact IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on the UniBody® that can bind to the cognate antigen (e.g., disease target), and thus the UniBody® binds monovalently to only one site on the target cell. For certain cancer cell surface antigens, this monovalent binding does not stimulate cancer cells to grow as may be seen using bivalent antibodies with the same antigen specificity, and thus UniBody® technology may offer a treatment option for some types of cancer that may be refractory to treatment with traditional antibodies. The small size of UniBody® may be a great advantage when treating some forms of cancer, allowing the molecule to be better distributed to larger solid tumors, possibly increasing efficacy.

[0117] In certain embodiments, the antibodies and antigen-binding fragments described herein are in the form of nanobodies. Minibodies are encoded by a single gene and have been efficiently produced in almost all prokaryotic and eukaryotic hosts, such as E. coli (see U.S. Pat. No. 6,765,087), molds (e.g., Aspergillus or Trichoderma), and yeasts (e.g., Saccharomyces, Kluyvermyces, Hansenula, or Pichia (see U.S. Pat. No. 6,838,254). The production process is scalable, and multi-kilogram quantities of nanobodies have been produced. Nanobodies can be formulated as ready-to-use liquids with long shelf lives. The Nanoclone method (see WO06 / 079372) is a proprietary method for generating nanobodies against desired targets based on automated high-throughput selection of B cells.

[0118] Also included are heavy chain dimers, such as antibodies from camels and sharks. Camel and shark antibodies contain two homodimeric pairs of chains of V-like and C-like domains (no light chains). The VH region of heavy chain dimeric IgG in camels does not need to make hydrophobic interactions with light chains, so the regions of the heavy chain that normally contact the light chains are changed to hydrophilic amino acid residues in camels. The VH domain of heavy chain dimeric IgG is called the VHH domain. Shark Ig-NAR contains a homodimer of one variable domain (called the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0119] In camels, the diverse antibody repertoire is determined by complementarity determining regions (CDRs) 1, 2, and 3 in the VH or VHH domains. CDR3 in camel VHH domains is characterized by a relatively long length of 16 amino acids on average (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This is in contrast to the CDRs of antibodies in many other species. In contrast to the R3 region. For example, the CDR3 of mouse VH has an average of 9 amino acids. A library of camel-derived antibody variable regions that maintains the in vivo diversity of camel variable regions can be generated, for example, by the methods disclosed in U.S. Patent Application Publication No. 20050037421, published February 17, 2005.

[0120] In certain embodiments, the antibody or antigen-binding fragment thereof is humanized. These embodiments refer to chimeric molecules, generally prepared using recombinant techniques, that have an antigen-binding site derived from an immunoglobulin of a non-human species and the remaining immunoglobulin structure of the molecule based on the structure and / or sequence of a human immunoglobulin. The antigen-binding site may comprise either a complete variable domain fused to a constant domain, or only the CDRs grafted into appropriate framework regions in the variable domain. The epitope-binding site may be wild-type or modified by one or more amino acid substitutions. This eliminates the constant region as an immunogen in human individuals, although the possibility of an immune response against the foreign variable region remains (LoBuglio et al., PNAS USA 86:4220-4224, 1989;Queen et al., PNAS USA. 86:10029-10033, 1988;Riechmann et al., Nature. 332:323-327, 1988). Exemplary methods for humanizing antibodies include those described in US Pat. No. 7,462,697.

[0121] Another approach focuses on not only providing constant regions of human origin, but also modifying the variable regions and reshaping them to be as close as possible to human form. It is known that both heavy and light chain variable regions contain three complementarity determining regions (CDRs) flanked by four framework regions (FRs) that are predicted to respond differently to the epitope in question, determine the binding ability, and are relatively conserved in a given species to provide a scaffold for the CDRs. When preparing a non-human antibody for a specific epitope, the variable region can be "reshaped" or "humanized" by grafting the CDRs from the non-human antibody onto the FRs present in the human antibody to be modified. Application of this approach to various antibodies has been reported in Sato et al., Cancer Res. 53:851-856, 1993;Riechmann et al., Nature 332:323-327, 1988;Verhoeyen et al., Science 239:1534-1536, 1988;Kettleborough et al., Protein Engineering. 4:773-3783, 1991;Maeda et al., Human Antibodies Hybridoma 2:124-134, 1991;Gorman et al., PNAS USA. 88:4181-4185, 1991;Tempest et al., Bio / Technology 9:266-271, 1991;Co et al., PNAS USA. 88:2869-2873, 1991; Carter et al., PNAS USA. 89:4285-4289, 1992; and Co et al., J Immunol. 148:1149-1154, 1992. In some embodiments, the humanized antibody preserves all CDR sequences (e.g., a humanized mouse antibody that contains all six CDRs from the mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (one, two, three, four, five, or six) that are altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody.

[0122] In certain embodiments, the antibody is a "chimeric" antibody. In this regard, a chimeric antibody is composed of an antigen-binding fragment of an antibody operably linked or otherwise fused to a heterologous Fc portion of a different antibody. In certain embodiments, the Fc domain or heterologous Fc domain is a domain of human origin. In certain embodiments, the Fc domain or heterologous Fc domain is a domain of mouse origin. In other embodiments, the heterologous Fc domain may be derived from a different Ig class from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In further embodiments, the heterologous Fc domain may be composed of CH2 and CH3 domains from one or more of the different Ig classes. As described above for humanized antibodies, an antigen-binding fragment of a chimeric antibody may include only one or more of the CDRs of an antibody described herein (e.g., one, two, three, four, five, or six CDRs of an antibody described herein) or may include the entire variable domain (VL, VH, or both).

[0123] It will be appreciated that any one or more of the foregoing IL-2 binding proteins can be combined with any of the other components described herein, such as IL-2 proteins, masking moieties comprising binding moieties and linkers, and other optional protein domains, to generate one or more activatable proproteins or larger multi-chain structures comprising the same.

[0124] Binding Moiety. As described above, the activatable proprotein homodimers described herein comprise a first polypeptide and a second polypeptide, each of which comprises a "binding moiety." The binding moiety facilitates and further stabilizes the binding interaction between the first and second polypeptides. In some embodiments, the binding moiety does not bind to IL-2 protein or to an IL-2 binding protein.

[0125] General examples of binding moieties are provided in Table M1 below. [Table M1]

[0126] Thus, in certain embodiments, the binding moiety is selected from Table M1.

[0127] In certain embodiments, the binding moiety comprises an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof, such as a VL domain and / or a VH domain. In some embodiments, the antigen-binding domain does not bind to an antigen, such as a human antigen. In some embodiments, the antigen-binding domain binds to an antigen, such as a human antigen.

[0128] In some embodiments, the binding moiety comprises a constant domain of an immunoglobulin, or a fragment or variant thereof. For example, in certain embodiments, the binding moiety comprises the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or CL domain of an immunoglobulin, including fragments and variants thereof, and combinations thereof. In some examples, the light chain (CL) is a lambda or kappa chain. In some embodiments, the constant domain present in the binding moiety of the activatable proprotein homodimer provided herein is glycosylated. In some embodiments, the glycosylation is N-glycosylation. In some embodiments, the glycosylation is O-glycosylation.

[0129] In specific embodiments, the binding moiety comprises, from N- to C-terminal: (1) an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof; and (2) an immunoglobulin constant domain, such as the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or CL domain of an immunoglobulin, including fragments and variants thereof. In specific embodiments, the binding moiety comprises, consists of, or consists essentially of the CH2CH3 domain of an immunoglobulin.

[0130] Immunoglobulin domains (antigen binding domains, constant domains) as used herein optionally include IgG domains. However, certain embodiments include alternative immunoglobulins, such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of various immunoglobulins are also encompassed by this embodiment. Thus, IgG1, IgG2, IgG3, etc. are all possible molecules in the binding domain. In addition to the choice of immunoglobulin type and isotype, certain embodiments include various hinge regions (or functional equivalents thereof). Such hinge regions provide flexibility between the different domains of the proproteins described herein. In some embodiments, the immunoglobulin portion of the binding domain (or larger masking portion) is derived from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgD, IgA, and IgM.

[0131] Linker. As described above, in certain embodiments, each polypeptide comprises at least one or at least two linkers or peptide linkers. In some embodiments, at least one of the linkers is a cleavable linker, for example, a cleavable linker that comprises a protease cleavage site. In some embodiments, at least one of the linkers is a non-cleavable linker, i.e., a physiologically stable linker.

[0132] In some embodiments, the first linker and / or the second linker is about 1-50, about 1-40, about 1-30, about 1-20, about 1-10, about 1-5, about 1-4, about 1-3 amino acids in length, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about The linker is about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 amino acids in length. In certain embodiments, the first linker is a cleavable linker and the second linker is a non-cleavable linker. In some embodiments, the first linker is a non-cleavable linker and the second linker is a cleavable linker. In some embodiments, both linkers are cleavable linkers.

[0133] In some embodiments, the cleavable linker comprises at least one protease cleavage site.Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., J. Gener. Virol. 78:699-722, 1997; and Scymczak et al., Nature Biotech. 5:589-594, 2004).In some embodiments, the protease cleavage site is cleavable by a protease selected from one or more of metalloproteases, serine proteases, cysteine ​​proteases, and aspartic acid proteases. In certain embodiments, the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A, and cathepsin B.

[0134] Examples of cleavable linkers are provided in Table S3 below. [Table S3-1] [Table S3-2] [Table S3-3]

[0135] Thus, in certain embodiments, the cleavable linker is selected from Table S3. Additional examples of cleavable linkers include amino acid sequences that are cleaved by serine proteases, such as thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin. Illustrative examples of amino acid sequences that are cleavable by thrombin include, but are not limited to: -Gly-Arg-Gly-Asp- (SEQ ID NO: 115), -Gly-Gly-Arg-, -Gly-Arg-Gly-Asp-Asn-Pro- (SEQ ID NO: 116), -Gly-Arg-Gly-Asp-Ser- (SEQ ID NO: 117), -Gly-Arg-Gly-Asp-Ser-Pro-Lys- (SEQ ID NO: 118), -Gly-Pro-Arg-, -Val-Pro-Arg-, and -Phe-Val-Arg-. Illustrative examples of amino acid sequences cleavable by an esterase include, but are not limited to: -Ala-Ala-Ala-, -Ala-Ala-Pro-Val- (SEQ ID NO: 119), -Ala-Ala-Pro-Leu- (SEQ ID NO: 120), -Ala-Ala-Pro-Phe- (SEQ ID NO: 121), -Ala-Ala-Pro-Ala- (SEQ ID NO: 122), and -Ala-Tyr-Leu-Val- (SEQ ID NO: 123).

[0136] Cleavable linkers also include amino acid sequences that can be cleaved by matrix metalloproteinases, such as collagenases, stromelysins, and gelatinases. Illustrative examples of amino acid sequences cleavable by matrix metalloproteinases include, but are not limited to: -Gly-Pro-Y-Gly-Pro-Z- (SEQ ID NO: 124), -Gly-Pro-, Leu-Gly-Pro-Z- (SEQ ID NO: 125), -Gly-Pro-Ile-Gly-Pro-Z- (SEQ ID NO: 126), and -Ala-Pro-Gly-Leu-Z- (SEQ ID NO: 127), where Y and Z are amino acids. Illustrative examples of amino acid sequences cleavable by collagenase include, but are not limited to: -Pro-Leu-Gly-Pro-D-Arg-Z- (SEQ ID NO: 128), -Pro-Leu-Gly-Leu-Leu-Gly-Z- (SEQ ID NO: 129), -Pro-Gln-Gly-Ile-Ala-Gly-Trp- (SEQ ID NO: 130), -Pro-Leu-Gly-Cys(Me)-His- (SEQ ID NO: 131), -Pro-Leu-Gly-Leu-Tyr-Ala- (SEQ ID NO: 132), -Pro-Leu-Ala-Leu-Trp-Ala-Arg- (SEQ ID NO: 133), and -Pro-Leu-Ala-Tyr-Trp-Ala-Arg- (SEQ ID NO: 134), where Z is an amino acid. An illustrative example of an amino acid sequence cleavable by stromelysin is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 135), and an example of an amino acid sequence cleavable by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 136).

[0137] Cleavable linkers also include amino acid sequences that can be cleaved by angiotensin converting enzyme, such as -Asp-Lys-Pro-, -Gly-Asp-Lys-Pro- (SEQ ID NO: 137), and -Gly-Ser-Asp-Lys-Pro- (SEQ ID NO: 138). Cleavable linkers also include amino acid sequences that can be degraded by cathepsin B, such as Val-Cit, Ala-Leu-Ala-Leu- (SEQ ID NO: 139), Gly-Phe-Leu-Gly- (SEQ ID NO: 140), and Phe-Lys.

[0138] In certain embodiments, the cleavable linker has a half-life at pH 7.4, 25° C., e.g., physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue) of about 30 minutes or less, about 1 hour or less, about 2 hours or less, about 3 hours or less, about 4 hours or less, about 5 hours or less, about 6 hours or less, about 12 hours or less, about 18 hours or less, about 24 hours or less, about 36 hours or less, about 48 hours or less, about 72 hours or less, or about 96 hours or less, or any half-life in between.

[0139] Typically, at least one of the first or second linker is a non-cleavable linker.Exemplary non-cleavable linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985;Murphy et al., PNAS USA. 83:8258-8262, 1986;US Pat. No. 4,935,233 and US Pat. No. 4,751,180.Particular non-cleavable linker sequences contain Gly, Ser, and / or Asn residues.Other near neutral amino acids, such as Thr and Ala, can also be used in peptide linker sequences if desired.

[0140] Certain exemplary non-cleavable linkers include Gly, Ser and / or Asn-containing linkers, as follows: [G] x, [S] x , [N] x , [GS] x , [GGS] x , [GSS] x , [GSGS] x (SEQ ID NO:141), [GGSG] x (SEQ ID NO:142), [GGGS] x (SEQ ID NO:143), [GGGGS] x (SEQ ID NO: 144), [GN] x , [GGN] x , [GNN] x , [GNGN] x (SEQ ID NO:145), [GGNG] x (SEQ ID NO:146), [GGGN] x (SEQ ID NO:147), [GGGGN] x (SEQ ID NO: 148) linker, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more. Other combinations of these and related amino acids will be apparent to one of skill in the art.

[0141] Additional examples of non-cleavable linkers include the following amino acid sequences: Gly-Gly-Gly-Gly-Gly-Ser-Gly ... ly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser- (SEQ ID NO: 151); Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-Ala-Ala-Ala-Ala-Arg-Asp-Ala-Ala-Ala-Lys- (SEQ ID NO: 152); and Asn-Val-Asp-His-Lys-Pro-Ser-Asn-Thr-Lys-Val-Asp-Lys-Arg- (SEQ ID NO: 153).

[0142] Further non-limiting examples of non-cleavable linkers include DGGGS (SEQ ID NO: 154); TGEKP (SEQ ID NO: 155) (see, e.g., Liu et al., PNAS. 94:5525-5530, 1997); GGRR (SEQ ID NO: 156) (Pomerantz et al. 1995); (GGGGS) n (SEQ ID NO:144) (Kim et al., PNAS. 93:1156-1160, 1996);EGKSSGSGSESKVD (SEQ ID NO:157) (Chaudhary et al., PNAS. 87:1066-1070, 1990);KESGSVSSEQLAQFRSLD (SEQ ID NO:158) (Bird et al., Science. 242:423-426, 1988),GGRRGGGS (SEQ ID NO:159);LRQRDGERP (SEQ ID NO:160);LRQKDGGGSERP (SEQ ID NO:161);LRQKd(GGGS) 2ERP (SEQ ID NO: 162). In a specific embodiment, the linker comprises a Gly3 linker sequence that comprises three glycine residues. In a specific embodiment, the flexible linker can be rationally designed using computer programs that can model both DNA binding sites and the peptide itself (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS. 91:11099-11103, 1994) or by phage display.

[0143] In some embodiments, the linker comprises an immunoglobulin (Ig) / antibody hinge region or fragment thereof, such as a hinge region obtained or derived from an IgG1 antibody. In some embodiments, the term Ig "hinge" region refers to a polypeptide comprising an amino acid sequence that shares sequence identity or similarity with a portion of a naturally occurring Ig hinge region sequence, optionally including cysteine ​​residues where disulfide bonds link the two heavy chains of the immunoglobulin. The sequence similarity between the hinge region linkers of the invention and naturally occurring immunoglobulin hinge region amino acid sequences can range from at least 50% to about 75-80%, and typically greater than about 90% similarity.

[0144] In some embodiments, the linker comprises a spacer element and a cleavable element to make the cleavable element more accessible to the enzyme responsible for cleavage.

[0145] It is understood that any one or more of the foregoing linkers can be combined with any one or more of the binding moieties, IL-2 proteins, IL-2 binding proteins and / or purification tags described herein to form an activatable proprotein homodimer of the present disclosure.

[0146] Affinity purification tags. In certain embodiments, the first and second polypeptides comprise at least one affinity purification tag, such as a polyhistidine tag (optionally a hexahistidine tag), a VSV-G tag (YTDIEMNRLGK; SEQ ID NO: 163), a universal tag (HTTPHH; SEQ ID NO: 164), a Strep tag (WSHPQFEK; SEQ ID NO: 165) or AWAHPQPGG; SEQ ID NO: 166), an S tag (KETAAAKFERQHMDS; SEQ ID NO: 167), an S1 tag (NANNPDWDF; SEQ ID NO: 168), a Phe tag ( For example, a Cys tag (e.g., comprised of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 Phe residues), a Cys tag (e.g., comprised of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 Cys residues), an Asp tag (e.g., comprised of about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, or about 12 Asp residues), an Arg tag (e.g., comprised of about 3, about 4, about 5, about about 6, about 7, about 8, about 9, about 10, about 11 or about 12 Arg residues), Myc epitope tag (CEQKLISEEDL, SEQ ID NO: 169), KT3 epitope tag (KPPTPPPEPET, SEQ ID NO: 170), HSV epitope tag (QPELAPED; SEQ ID NO: 171), histidine affinity tag (KDHLIHNVHKEFHAHAHNK; SEQ ID NO: 172), hemagglutinin (HA) tag, Exemplary affinity purification tags include a FLAG epitope tag (DYKDDDK; SEQ ID NO: 173), an E2 epitope tag (SSTSSDFRDR; SEQ ID NO: 174), a V5 tag (GKPIPNPLLGLDST; SEQ ID NO: 175), a T7 tag (MASMTGGQQMG; SEQ ID NO: 176), an AU5 epitope tag (TDFYLK; SEQ ID NO: 177), and an AU1 epitope tag (DTYRYI; SEQ ID NO: 178).

[0147] Additional domains. Certain activatable proproteins contain one or more additional domains, such as binding domains. In some embodiments, each of the polypeptides in the activatable proprotein further comprises protein domain A at one free end and / or protein domain B at the other free end.

[0148] In some embodiments, protein domains A and B are the same or different. In certain embodiments, protein domains A and B are selected from one or more of a cell receptor targeting moiety, optionally a bispecific targeting moiety, an antigen binding domain, optionally a bispecific antigen binding domain, a cell membrane receptor extracellular domain (ECD), an Fc domain, human serum albumin (HSA), an Fc binding domain, an HSA binding domain, a cytokine, a chemokine, and a soluble protein ligand.

[0149] In some embodiments, one or more additional protein domains can be used to form complexes of two, three, four, five, or more activatable proproteins, which bind to each other via the additional domain(s).

[0150] Illustrative examples of activatable proproteins and some of their predicted cleavage products are provided in Table S4 below (see also Examples). [Table S4-1] [Table S4-2] [Table S4-3] [Table S4-4] [Table S4-5] [Table S4-6] [Table S4-7]

Table S4-8

Table S4-9

Table S4-10

Table S4-11

Table S4-12

Table S4-13

Table S4-14

Table S4-15

Table S4-16

Table S4-17

Table S4-18

Table S4-19

Table S4-20

Table S4-21

Table S4-22

Table S4-23

Table S4-24

Table S4-25

Table S4-26

Table S4-27

Table S4-28

Table S4-29

Table S4-30

Table S4-31

Table S4-32

Table S4-33

Table S4-34

Table S4-35

Table S4-36

Table S4-37

Table S4-38

Table S4-39

Table S4-40

Table S4-41

Table S4-42

Table S4-43

Table S4-44

Table S4-45

Table S4-46

Table S4-47

Table S4-48

[0151] Thus, in certain embodiments, an activatable proprotein comprises a first polypeptide comprising, consisting of, or consisting essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a sequence selected from Table S4. In certain embodiments, the protease cleavage site (e.g., a TEV protease cleavage site) for any one or more of the foregoing sequences (from Table S4) is replaced with a human protease cleavage site, i.e., a cleavage site cleavable by a human protease, e.g., a human protease expressed in cancer tissue or cancer cells (see, e.g., Table S3 for exemplary cleavable linkers).

[0152] Methods of Use and Pharmaceutical Compositions Certain embodiments include a method of treating, alleviating symptoms, and / or reducing the progression of a disease or condition in a subject in need thereof, comprising administering to the subject at least one activatable proprotein described herein. Also included is a method of enhancing an immune response in a subject, comprising administering to the subject at least one activatable proprotein described herein. In certain embodiments, the disease is selected from one or more of cancer, viral infection, and immune disorder.

[0153] In some embodiments, after administration, the activatable proprotein is activated through protease cleavage in cells or tissues, releasing or opening homodimers and exposing a binding site for the IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo, thereby generating an activated protein (see, e.g., Figures 4A-4D). In certain embodiments, the protease cleavage occurs in cancer cells or tissues, or in virus-infected cells or tissues. Typically, the activated protein has at least one immunostimulatory IL-2 activity, for example, by binding to the IL-2Rβ / γc chain present on the surface of immune cells in vivo, and thereby stimulating the immune cells. In certain embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0154] In some embodiments, administration and activation of an activatable proprotein to generate an activated protein increases an immune response in a subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, compared to a control. In some examples, the immune response is an anti-cancer or anti-viral immune response. In some embodiments, administration and activation of an activatable proprotein to generate an activated protein increases cell killing in a subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, compared to a control.In some embodiments, the cell killing is the killing of cancer cells or the killing of virally infected cells.

[0155] In some embodiments, administration and activation of an activatable proprotein to generate an activator protein is inducible by the activation of regulatory T cells (T reg (e.g., the disulfide bond between the IL-2 protein and the IL-2Rα protein) is maintained after cleavage of the linker, and the binding of the activator protein to the IL-2Rα / β / γc chain expressed on T reg IL-2Rα / β / γc chains expressed on T reg Thus, in certain embodiments, the activator protein has a higher binding affinity than the activatable proprotein (T reg ) proliferation and / or activation.

[0156] In some embodiments, the disease is cancer, i.e. the subject in need thereof has or is suspected of having cancer.Therefore, certain embodiments include a method of treating cancer, alleviating symptoms of cancer, or inhibiting the progression of cancer in a subject in need thereof, comprising administering to the subject at least one activatable proprotein described herein.In certain embodiments, the cancer is primary cancer or metastatic cancer. In specific embodiments, the cancer is selected from one or more of melanoma (optionally metastatic melanoma), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer.

[0157] In some embodiments, as described above, cancer is metastatic cancer.In addition to the above cancer, exemplary metastatic cancer includes, but is not limited to, bladder cancer that metastasizes to bone, liver, and / or lung; breast cancer that metastasizes to bone, brain, liver, and / or lung; colorectal cancer that metastasizes to liver, lung, and / or peritoneum; kidney cancer that metastasizes to adrenal gland, bone, brain, liver, and / or lung; lung cancer that metastasizes to other parts of adrenal gland, bone, brain, liver, and / or lung; melanoma that metastasizes to bone, brain, liver, lung, and / or skin / muscle; ovarian cancer that metastasizes to liver, lung, and / or peritoneum; pancreatic cancer that metastasizes to liver, lung, and / or peritoneum; prostate cancer that metastasizes to adrenal gland, bone, liver, and / or lung; gastric cancer that metastasizes to liver, lung, and / or peritoneum; thyroid cancer that metastasizes to bone, liver, and / or lung; and uterine cancer that metastasizes to bone, liver, lung, peritoneum, and / or vagina.

[0158] The method of treating cancer can be combined with other therapeutic modalities.For example, the combination therapy described herein can be administered to a subject before, during, or after other therapeutic interventions, including symptomatic care, radiation therapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof.Symptomatic care includes administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and administration of anticonvulsants to reduce seizures.Radiation therapy includes whole brain irradiation, fractionated radiation therapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0159] Thus, certain embodiments include combination therapies for treating cancer, including methods of treating, alleviating symptoms of, or inhibiting progression of cancer in a subject in need thereof, comprising administering at least one activatable proprotein as described herein in combination with at least one additional agent, such as a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor. In some embodiments, administering at least one activatable proprotein increases the sensitivity of the cancer to the additional agent (e.g., a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor) by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 150%, about or at least about 200%, about or at least about 250%, about or at least about 300%, about or at least about 350%, about or at least about 400%, about or at least about 450%, about or at least about 50 ...00%, about or at least about 200%, about or at least about 200%, about or at Increase by about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more.

[0160] Certain combination therapies employ one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents, non-limiting examples of which include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or type II), anti-microtubule agents, among others.

[0161] Examples of alkylating agents include nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, mustine, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolomide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and nonclassical alkylating agents (procarbazine and hexamethylmelamine, as appropriate).

[0162] Examples of antimetabolites include antifolates (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, enocitabine, cytarabine, gemcitabine, decitabine, azacytidine, fludarabine, nelarabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0163] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0164] Examples of anti-microtubule agents include the taxanes (eg, paclitaxel and docetaxel) and the vinca alkaloids (eg, vinblastine, vincristine, vindesine, vinorelbine).

[0165] It will be understood by those of skill in the art that the various chemotherapeutic agents described herein can be combined with any one or more of the activatable proproteins described herein and used in accordance with any one or more of the methods or compositions described herein.

[0166] Certain combination therapy uses at least one hormone therapy agent.General examples of hormone therapy agents include hormone agonists and hormone antagonists.Specific examples of hormone agonists include progestogens (progestins), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factors, VEGF-derived angiogenic and lymphangiogenic factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galectin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors, including their analogs, and gonadotropin releasing hormone (GnRH) agonists (e.g., leuprolide, goserelin, triptorelin, histrelin).Similarly, examples of hormone receptor antagonists include selective estrogen receptor modulators (SERMs; e.g., tamoxifen, raloxifene, toremifene) and antiandrogens (e.g., flutamide, bicalutamide, nilutamide).

[0167] Also included are hormone pathway inhibitors, such as antibodies against hormone receptors. Examples include inhibitors of the IGF receptor (e.g., IGF-IR1), such as cixutumumab, dalotuzumab, figitumumab, ganitumab, istiratumab, and lobatumumab; inhibitors of vascular endothelial growth factor receptor 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab pegol, bevacizumab, icrucumab, and ramucirumab; inhibitors of TGF-beta receptor R1, R2, and R3, such as fresolimumab and methelimumab; c-M inhibitors of EGF receptors, such as cetuximab, depatuxizumab mafodotin, futuximab, imgatuzumab, laprituximab emtansine, matuzumab, modotuximab, necitumumab, nimotuzumab, panitumumab, tomzotuximab, and zalutumumab; inhibitors of FGF receptors, such as aprtumab ixadotin and bemarituzumab; and inhibitors of PDGF receptors, such as olaratumab and tobetumab.

[0168] It will be understood by those of skill in the art that the various hormonal therapy agents described herein can be combined with any one or more of the various activatable proproteins described herein and used in accordance with any one or more of the methods or compositions described herein.

[0169] Certain combination therapies use at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, but are not limited to, adavosertib, afantinib, aflibercept, axitinib, bevacizumab, bosutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, These include ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranibizumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, trastuzumab, vandetanib, and vemurafenib.

[0170] It will be understood by those of skill in the art that the various kinase inhibitors described herein can be combined with any one or more of the various activatable proproteins described herein and used in accordance with any one or more of the methods or compositions described herein.

[0171] In some embodiments, the methods and pharmaceutical compositions described herein extend the median survival time of subjects by 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 15 weeks, 20 weeks, 25 weeks, 30 weeks, 40 weeks or more.In certain embodiments, the methods and pharmaceutical compositions described herein extend the median survival time of subjects by 1 year, 2 years, 3 years or more.In some embodiments, the methods and pharmaceutical compositions extend progression-free survival by 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks or more.In certain embodiments, the methods and pharmaceutical compositions described herein extend progression-free survival by 1 year, 2 years, 3 years or more.

[0172] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause a statistically significant reduction in the amount of viable tumor, such as a reduction in tumor mass of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or more, or tumor regression as indicated by altered (e.g., statistically significantly reduced) scan dimensions. In certain embodiments, the methods and therapeutic compositions described herein are sufficient to cause stable disease.

[0173] In some embodiments, the disease is a viral disease or viral infection.In certain embodiments, the viral infection is selected from one or more of the following: human immunodeficiency virus (HIV), hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis E virus, calicivirus-associated diarrhea, rotavirus diarrhea, Haemophilus influenzae type B pneumonia and invasive disease, influenza, measles, mumps, rubella, parainfluenza-associated pneumonia, respiratory syncytial virus (RSV) pneumonia, severe acute respiratory syndrome (SARS), human papillomavirus, herpes simplex type 2 genital ulcer, dengue fever, Japanese encephalitis, tick-borne encephalitis, West Nile virus-associated disease, yellow fever, Epstein-Barr virus, Lassa fever, Crimean-Congo hemorrhagic fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, rabies, Rift Valley fever, smallpox, upper and lower respiratory tract infections, and poliomyelitis.In a specific embodiment, the subject is HIV positive. In some embodiments, the methods and pharmaceutical compositions described herein increase an anti-viral immune response by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, as compared to a control.

[0174] In some embodiments, the immune disorder is selected from one or more of type 1 diabetes, vasculitis, and immunodeficiency. In some embodiments, the methods and pharmaceutical compositions described herein improve immune function in a subject by, e.g., about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, as compared to a control.

[0175] In certain embodiments, the methods and therapeutic compositions described herein are sufficient to result in a clinically relevant reduction in the symptoms of a particular disease indication known to a skilled clinician.

[0176] As noted above, when used in vivo for the treatment or testing of human or non-human mammalian disease, the agents described herein are typically incorporated into one or more therapeutic or pharmaceutical compositions, including veterinary therapeutic compositions, prior to administration.

[0177] Thus, certain embodiments relate to pharmaceutical or therapeutic compositions comprising at least one activatable proprotein as described herein. In some examples, the pharmaceutical or therapeutic compositions comprise one or more of the activatable proproteins as described herein in combination with a pharma- ceutical or physiologically acceptable carrier or excipient. Certain pharmaceutical or therapeutic compositions further comprise at least one additional agent, such as a chemotherapeutic agent, a hormonal therapy agent, and / or a kinase inhibitor as described herein.

[0178] Some therapeutic compositions include (and certain methods utilize) only one activatable proprotein. Certain therapeutic compositions include (and certain methods utilize) a mixture of at least two, at least three, at least four, or at least five different activatable proproteins.

[0179] In certain embodiments, pharmaceutical or therapeutic compositions comprising at least one activatable proprotein are substantially pure on a protein basis or on a weight-by-weight basis, e.g., the compositions have a purity of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% on a protein basis or on a weight-by-weight basis.

[0180] In certain embodiments, the first and second polypeptides prior to cleavage are substantially in homodimeric form in a composition or other physiological solution or under physiological conditions, e.g., in vivo conditions.

[0181] In some embodiments, the activatable proproteins described herein do not form aggregates as known in the art, have a desired solubility, and / or have an immunogenicity profile suitable for use in humans. Thus, in some embodiments, a therapeutic composition comprising an activatable proprotein is substantially free of aggregates. For example, certain compositions comprise less than about 10% (on a protein basis) high molecular weight aggregated protein, or less than about 5% high molecular weight aggregated protein, or less than about 4% high molecular weight aggregated protein, or less than about 3% high molecular weight aggregated protein, or less than about 2% high molecular weight aggregated protein, or less than about 1% high molecular weight aggregated protein. Some compositions comprise an activatable proprotein that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% monodisperse with respect to its apparent molecular mass.

[0182] In some embodiments, the activatable proprotein is concentrated to about or at least about 0.1 mg / ml, about or at least about 0.2 mg / ml, about or at least about 0.3 mg / ml, about or at least about 0.4 mg / ml, about or at least about 0.5 mg / ml, about or at least about 0.6, about or at least about 0.7, about or at least about 0.8, about or at least about 0.9, about or at least about 1 mg / ml, about or at least about 2 mg / ml, about or at least about 3 mg / ml, about or at least about 4 mg / ml, about or at least about 5 mg / ml, about or at least about 6 mg / ml, about or at least about 7 mg / ml, about or at least about 8 mg / ml, about or at least about 9 mg / ml, about or at least about 10 mg / ml, about or at least about 11, about or at least about 12, about or at least about 13, about or at least about 14, or about or at least about 15 mg / ml and formulated for biotherapeutic use.

[0183] To prepare a therapeutic or pharmaceutical composition, an effective or desired amount of one or more agents is mixed with any pharmaceutical carrier(s) or excipients known to those skilled in the art to be suitable for a particular agent and / or mode of administration. Pharmaceutical carriers can be liquid, semi-liquid, or solid. The solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include, for example, sterile diluents (e.g., water), saline solutions (e.g., phosphate buffered saline; PBS), fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents (e.g., benzyl alcohol and methylparabens); antioxidants (e.g., ascorbic acid and sodium bisulfite), and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); buffers (e.g., acetates, citrates, and phosphates). If administered intravenously (eg, by IV infusion), suitable carriers include saline or phosphate buffered saline (PBS), as well as solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0184] Administration of the agents described herein in pure form or in a suitable therapeutic or pharmaceutical composition can be carried out via any of the accepted modes of administration of the agent to accomplish similar utilities. Therapeutic or pharmaceutical compositions can be prepared by combining the agent-containing composition with a suitable physiologically acceptable carrier, diluent, or excipient, and may be formulated into solid, semi-solid, liquid, or gaseous preparations, such as tablets, capsules, powders, granules, ointments, liquids, suppositories, injections, inhalants, gels, microspheres, and aerosols. In addition, other pharmacologic active ingredients (including other small molecules described elsewhere herein) and / or suitable excipients, such as salts, buffers, and stabilizers, may also be present in the composition, but are not necessarily present.

[0185] Administration can be accomplished by a variety of routes, including oral, parenteral, nasal, intravenous, intradermal, intramuscular, subcutaneous, or topical. The preferred mode of administration depends on the nature of the condition to be treated or prevented. Certain embodiments include administration by IV infusion.

[0186] A carrier can include, for example, a pharma- ceutically or physiologically acceptable carrier, excipient, or stabilizer that is not toxic to cells or mammals exposed thereto at the dosages and concentrations used. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or non-ionic surfactants such as polysorbate 20 (TWEEN®), polyethylene glycol (PEG), and poloxamer (PLURONICS®), and the like.

[0187] In some embodiments, one or more agents can be entrapped in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions, prepared, for example, by coacervation techniques or interfacial polymerization. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may further comprise other therapeutic or diagnostic agents.

[0188] The exact dosage and duration of treatment are a function of the disease being treated and can be determined empirically using known test protocols or by testing the composition in model systems known in the art and extrapolating therefrom. Comparative clinical trials can also be carried out. Dosage can also vary according to the severity of the condition being alleviated. Pharmaceutical compositions are generally formulated and administered to exert a therapeutically useful effect while minimizing undesirable side effects. The composition can be administered once or divided into several smaller doses that are administered at intervals of time. For any particular subject, the specific dosage regimen can be adjusted over time according to the individual's needs.

[0189] Typical routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection or infusion techniques. The therapeutic or pharmaceutical compositions according to certain embodiments of the present disclosure are formulated so that the active ingredients contained therein are bioavailable upon administration of the composition to a subject or patient. The composition administered to a subject or patient may take the form of one or more dosage units, e.g., a tablet may be a single dosage unit, and a container of an agent described herein in aerosol form may hold multiple dosage units. The actual methods of preparing such dosage forms are known or apparent to those skilled in the art, see, for example, Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition administered typically contains a therapeutically effective amount of an agent described herein for the treatment of the disease or condition of interest.

[0190] The therapeutic or pharmaceutical composition may be in solid or liquid form. In one embodiment, the carrier(s) are particulate, so that the composition is, for example, in tablet or powder form. The carrier(s) may be liquid, in which case the composition is, for example, an oral oil, an injectable liquid or an aerosol, which is useful, for example, in inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, with semi-solid, semi-liquid, suspension, and gel forms being included among the forms discussed herein as either solid or liquid. Certain embodiments include sterile injectable solutions.

[0191] As a solid composition for oral administration, the pharmaceutical composition may be formulated into powders, granules, compressed tablets, pills, capsules, gums, wafers, and the like. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethylcellulose, ethylcellulose, microcrystalline cellulose, tragacanth, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginic acid, sodium alginate, Primogel, corn starch, and the like; lubricants, such as magnesium stearate, or Sterotex; lubricants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; flavorings, such as peppermint, methyl salicylate, or orange flavor; and coloring agents. When the pharmaceutical composition is in the form of a capsule, such as a gelatin capsule, it may contain, in addition to materials of the above types, a liquid carrier, such as polyethylene glycol or oil.

[0192] The therapeutic or pharmaceutical composition can be in liquid form, for example, elixir, syrup, solution, emulsion, or suspension. The liquid can be for oral administration or for delivery by injection, as two examples. When intended for oral administration, the preferred composition contains one or more of sweeteners, preservatives, dyes / colorants, and flavor enhancers in addition to the compound of the present invention. In the composition intended for administration by injection, one or more of surfactants, preservatives, wetting agents, dispersing agents, suspending agents, buffers, stabilizers, and isotonic agents can be included.

[0193] Liquid therapeutic or pharmaceutical compositions, whether they are in solution, suspension, or other similar form, may contain one or more of the following auxiliary agents: sterile diluents, such as water for injection, saline solution, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils, such as synthetic mono- or diglycerides, polyethylene glycols, glycerin, propylene glycol, or other solvents that may serve as solvents or suspending media; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite, chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates, and agents for adjusting isotonicity, such as sodium chloride or dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Saline is the preferred auxiliary agent. Injectable pharmaceutical compositions are preferably sterile.

[0194] Liquid therapeutic or pharmaceutical compositions intended for either parenteral or oral administration should contain an amount of drug such that a suitable dosage is obtained. Typically, this amount is at least 0.01% of the drug of interest in the composition. If intended for oral administration, this amount may vary between 0.1 and about 70% by weight of the composition. Certain oral therapeutic or pharmaceutical compositions contain about 4% to about 75% of the drug of interest. In certain embodiments, therapeutic or pharmaceutical compositions and preparations are prepared such that a parenteral dosage unit contains 0.01 to 10% by weight of the drug of interest before dilution.

[0195] The therapeutic or pharmaceutical composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base.For example, the base may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers.Thickeners may be present in the therapeutic or pharmaceutical composition for topical administration.When intended for transdermal administration, the composition may comprise a transdermal patch or iontophoresis device.

[0196] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories that melt in the rectum and release the drug.Compositions for rectal administration may contain an oily base as a suitable non-irritating excipient.Such bases include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0197] Therapeutic or pharmaceutical compositions may contain various materials that modify the physical form of solid or liquid dosage units. For example, the composition may contain materials that form a coating shell around the active ingredient. The materials that form the coating shell are typically inert and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredient may be encapsulated in a gelatin capsule. Therapeutic or pharmaceutical compositions in solid or liquid form may contain components that bind to the agent and thereby aid in the delivery of the compound. Suitable components that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0198] The therapeutic or pharmaceutical composition may consist essentially of a dosage unit that can be administered as an aerosol. The term aerosol is used to refer to a variety of systems ranging from systems of colloidal nature to systems consisting of pressurized packaging. Delivery may be by liquefied or pressurized gas or by a suitable pump system that dispenses the active ingredient. The aerosol may be delivered in a single-phase, two-phase, or three-phase system to deliver the active ingredient(s). Delivery of the aerosol includes the necessary containers, activators, valves, subcontainers, etc., which may together form a kit. Those skilled in the art may determine the preferred aerosol without undue experimentation.

[0199] The compositions described herein can be prepared with carriers that protect the agent against rapid elimination from the body, such as sustained release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and others known to those skilled in the art.

[0200] Therapeutic or pharmaceutical compositions can be prepared by methodology well known in the pharmaceutical art. For example, therapeutic or pharmaceutical compositions intended to be administered by injection can contain one or more of salts, buffers, and / or stabilizers together with sterile distilled water to form a solution. Surfactants may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that interact with agents non-covalently to facilitate the dissolution or homogeneous suspension of the agents in aqueous delivery systems.

[0201] The therapeutic or pharmaceutical composition may be administered in a therapeutically effective amount, which varies depending on a variety of factors, including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the age, weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject being treated. In some examples, the therapeutically effective daily dose is about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g) (for a 70 kg mammal); preferably, the therapeutically effective dose is about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., about 3.5 g) (for a 70 kg mammal); more preferably, the therapeutically effective dose is about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g) (for a 70 kg mammal). In some embodiments, the therapeutically effective dose is administered weekly, biweekly, or monthly. In specific embodiments, a therapeutically effective dose is administered weekly, biweekly, or monthly, for example at a dose of about 1-10 mg / kg or about 1-5 mg / kg, or about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, or about 10 mg / kg.

[0202] Combination therapy as described herein may include administration of a single pharmaceutical dosage formulation containing an activatable proprotein and an additional therapeutic agent (e.g., a chemotherapeutic agent, a hormonal therapy agent, a kinase inhibitor), as well as administration of a composition containing an activatable proprotein and an additional therapeutic agent in its own separate pharmaceutical dosage formulation. For example, the activatable proprotein and the additional therapeutic agent can be administered to the subject together in a single oral dosage composition, such as a tablet or capsule, or each agent can be administered in a separate oral dosage formulation. Similarly, the activatable proprotein and the additional therapeutic agent can be administered to the subject together in a single parenteral dosage composition, such as a saline solution or other physiologically acceptable solution, or each agent can be administered in a separate parenteral dosage formulation. As another example, with respect to cell-based therapy, the activatable proprotein can be mixed with the cells prior to administration, administered as part of a separate composition, or both. When separate dosage formulations are used, the compositions can be administered at essentially the same time, i.e., together, or individually at alternating times, i.e., sequentially and in any order; combination therapy is understood to include all of these regimens.

[0203] Also included are patient care kits that include (a) at least one activatable proprotein described herein; and, optionally, (b) at least one additional agent (e.g., a chemotherapeutic agent, a hormonal therapy agent, a kinase inhibitor). In certain kits, (a) and (b) are present in separate therapeutic compositions. In some kits, (a) and (b) are present in the same therapeutic composition.

[0204] The kits described herein may also include one or more additional therapeutic agents or other components suitable or desirable for the indication being treated or for the desired diagnostic use. The kits described herein may also include one or more syringes or other components (e.g., stents, implantable depots, etc.) necessary or desirable to facilitate the intended mode of delivery.

[0205] In some embodiments, the patient care kit contains separate containers, dividers, or compartments for the composition(s) and the informational material(s). For example, the composition(s) can be contained in a bottle, vial, or syringe, and the informational material(s) can be contained in association with the container. In some embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe to which the informational material is attached in the form of a label. In some embodiments, the kit contains a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., dosage forms described herein) of the activatable proprotein and, optionally, at least one additional therapeutic agent. For example, the kit contains a plurality of syringes, ampoules, foil packets, or blister packs, each containing a single unit dose of the activatable proprotein and, optionally, at least one additional therapeutic agent. The containers of the kit can be airtight, waterproof (e.g., impermeable to changes in moisture or evaporation), and / or light-tight.

[0206] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, an inhaler, a dropper (e.g., an eye dropper), a swab (e.g., a cotton swab or a wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a fixed dose of the agent(s). Also included are methods of providing the kit, such as by combining the components described herein.

[0207] Expression and purification system Certain embodiments include methods and related compositions for expressing and purifying the activatable proproteins described herein. Such recombinant activatable proproteins can be conveniently prepared using standard protocols, for example as described in Sambrook, et al., (1989, supra), especially chapters 16 and 17; Ausubel et al., (1994, supra), especially chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995-1997), especially chapters 1, 5 and 6. As one general example, activatable proproteins can be prepared by a procedure that includes one or more of the following steps: (a) preparing one or more vectors or constructs that include one or more polynucleotide sequences encoding the individual polypeptide chains of a homodimer operably linked to one or more regulatory elements; (b) introducing the one or more vectors or constructs into one or more host cells; (c) culturing the one or more host cells to express the polypeptides that combine with each other to form the activatable proprotein homodimer; and (d) isolating the activatable proprotein homodimer from the host cells. Alternatively, the polypeptide chains can first be isolated and produced in the host cells and then incubated under suitable conditions to form the activatable proprotein homodimer.

[0208] To express the desired polypeptide, the nucleotide sequence encoding the first and / or second polypeptide chain of the activatable proprotein may be inserted into a suitable expression vector, i.e., a vector containing the necessary elements for the transcription and translation of the inserted coding sequence. Methods well known to those skilled in the art may be used to construct an expression vector containing a sequence encoding the desired polypeptide and appropriate transcription and translation control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in Sambrook et al., Molecular Cloning, A Laboratory Manual (1989), and Ausubel et al., Current Protocols in Molecular Biology (1989).

[0209] A variety of expression vector / host systems are known and can be utilized to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms, such as bacteria transformed by recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed by yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculovirus); plant cell systems transformed by viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or by bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems, including mammalian cells and more specifically human cell systems.

[0210] The "control elements" or "regulatory sequences" present in an expression vector are the untranslated regions of the vector that interact with host cell proteins to carry out transcription and translation, i.e. enhancers, promoters, 5' and 3' untranslated regions. Such elements may vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements may be used, including constitutive and inducible promoters. For example, when cloning in bacterial systems, inducible promoters may be used, such as the hybrid lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) or PSPORT1 plasmid (Gibco BRL, Gaithersburg, Md.). In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are generally preferred. When it is necessary to generate a cell line containing multiple copies of a polypeptide-encoding sequence, vectors based on SV40 or EBV may be conveniently used with an appropriate selectable marker.

[0211] In bacterial systems, several expression vectors may be selected depending on the intended use of the expressed polypeptide. For example, if large quantities are required, vectors directing high-level expression of fusion proteins that are easily purified may be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which a sequence encoding a polypeptide of interest may be ligated into the vector in frame with the sequence of the amino-terminal Met followed by the seven residues of β-galactosidase to produce a hybrid protein; pIN vectors (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)), and the like. pGEX vectors (Promega, Madison, Wis.) may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. Proteins made in such systems may be designed to contain heparin, thrombin, or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.

[0212] Certain embodiments use an E. coli-based expression system (see, e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146, 2008). These and related embodiments may rely partially or completely on ligation-independent cloning (LIC) to generate suitable expression vectors. In specific embodiments, protein expression may be controlled by T7 RNA polymerase (e.g., the pET vector series). These and related embodiments may utilize the expression host strain BL21(DE3), a lambda DE3 lysogen of BL21 that supports T7-mediated expression and is deficient in lon and ompT proteases to improve target protein stability. Also included are expression host strains with plasmids encoding tRNAs rarely used in E. coli, such as the ROSETTA™(DE3) and Rosetta2(DE3) strains. Cell lysis and sample handling may also be improved using reagents sold under the trademarks BENZONASE® Nuclease and BUGBUSTER® Protein Extraction Reagent. With respect to cell culture, autoinduction media can improve the efficiency of many expression systems, including high-throughput expression systems. This type of media (e.g., the OVERNIGHT EXPRESS™ Autoinduction System) gradually induces protein expression through a metabolic shift without the addition of artificial inducers such as IPTG. Certain embodiments use a hexahistidine tag (e.g., a tag sold under the trademark HIS·TAG® fusion) followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in certain aspects, clinical grade proteins can be isolated from E. coli inclusion bodies without or without the use of affinity tags (see, e.g., Shimp et al., Protein Expr Purif. 50:58-67, 2006).As a further example, certain embodiments may use a cold-shock-inducible E. coli high-yield production system, as overexpression of a protein in Escherichia coli at low temperatures improves its solubility and stability (see, e.g., Qing et al., Nature Biotechnology. 22:877-882, 2004).

[0213] Similarly, high-density bacterial fermentation systems are included. For example, high cell density cultures of Ralstonia eutropha allow protein production at cell densities of over 150 g / L, and recombinant proteins have been expressed at titers of over 10 g / L.

[0214] In yeast Saccharomyces cerevisiae, several vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH, may be used. For reviews, see Ausubel et al. (supra) and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included are Pichia pandoris expression systems (see, for example, Li et al., Nature Biotechnology. 24, 210-215, 2006; and Hamilton et al., Science, 301:1244, 2003). Certain embodiments include yeast systems engineered to selectively glycosylate proteins, including yeast with humanized N-glycosylation pathways, among others (see, e.g., Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., Nature Reviews Microbiol. 3:119-28, 2005; and Gerngross et al., Nature-Biotechnology. 22:1409 -1414, 2004; U.S. Patent Nos. 7,629,163; 7,326,681; and 7,029,872). By way of example only, recombinant yeast cultures can be grown in Fernbach flasks or 15L, 50L, 100L, and 200L fermentors, among others.

[0215] When using plant expression vectors, the expression of the sequence encoding the polypeptide can be driven by any of several promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with the omega leader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)). Alternatively, plant promoters such as the small subunit or heat shock promoter of RUBISCO can be used (Coruzzi et al., EMBO J. 3:1671-1680 (1984); Broglie et al., Science 224:838-843 (1984); and Winter et al., Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection. Such techniques are described in several generally available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0216] Insect systems can also be used to express polypeptides of interest. For example, in one such system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes in Spodoptera frugiperda cells or Trichoplusia cells. The sequence coding for the polypeptide is cloned into a non-essential region of the virus, such as the polyhedrin gene, and placed under the control of the polyhedrin promoter. If the polypeptide coding sequence is successfully inserted, the polyhedrin gene is inactivated, producing a recombinant virus that lacks coat protein. The recombinant virus can then be used to infect, for example, S.frugiperda cells or Trichoplusia cells to express the polypeptide of interest (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)). Also included are baculovirus expression systems, including systems utilizing SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5:Unit5.4, 2001). Insect systems can provide post-translational modifications similar to mammalian systems.

[0217] In mammalian host cells, several virus-based expression systems are commonly available. For example, when adenovirus is used as an expression vector, the sequence encoding the polypeptide of interest may be ligated into the adenovirus transcription / translation complex consisting of the late promoter and tripartite leader sequence. Insertion in the non-essential E1 or E3 region of the viral genome may be used to obtain a viable virus capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). In addition, transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.

[0218] Examples of useful mammalian host cell lines include the SV40 transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC5 cells; FS4 cells; and human hepatoma lines (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNAS USA 77:4216 (1980)); and myeloma cell lines, such as NSO and Sp2 / 0. For a review of certain mammalian host cell systems suitable for protein production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 255-268. Certain preferred mammalian cell expression systems include CHO and HEK293 cell-based expression systems.Mammalian expression systems can utilize adherent cell lines, e.g., in T-flasks, roller bottles, or cell factories, or suspension cultures, e.g., in 1 L and 5 L spinners, 5 L, 14 L, 40 L, 100 L, and 200 L stirred tank bioreactors, or 20 / 50 L and 100 / 200 L WAVE bioreactors, among others known in the art.

[0219] Also included is cell-free expression of proteins. These and related embodiments typically utilize purified RNA polymerase, ribosomes, tRNA and ribonucleotides; these reagents may be produced by extraction from cells or from cell-based expression systems.

[0220] Specific initiation signals can also be used to achieve more efficient translation of sequences encoding polypeptides of interest. Such signals include the ATG initiation codon and adjacent sequences. When the sequence encoding a polypeptide, its initiation codon, and upstream sequences are inserted into a suitable expression vector, no additional transcriptional or translational control signals are required. However, when only a coding sequence or a portion thereof is inserted, exogenous translational control signals including the ATG initiation codon should be provided. Furthermore, the initiation codon should be in the correct reading frame to ensure translation of the entire insert. Exogenous translational elements and initiation codons can be of various origins, both natural and synthetic. Expression efficiency can be enhanced by including enhancers that are appropriate for the particular cell system used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0221] In addition, a host cell line may be selected for its ability to modulate the expression of inserted sequences or to process expressed proteins in a desired manner. Such modifications of polypeptides include, but are not limited to, post-translational modifications, such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing that cleaves the "prepro" form of a protein may also be used to facilitate correct insertion, folding, and / or function. In addition to bacterial cells, different host cells, such as yeast, CHO, HeLa, MDCK, HEK293, and W138, that have or lack specific cellular machinery and characteristic mechanisms for such post-translational activity, may be selected to ensure the correct modification and processing of foreign proteins.

[0222] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines stably expressing a polynucleotide of interest may be transformed using expression vectors that may contain a viral origin of replication and / or endogenous expression elements and a selectable marker gene on the same or a separate vector. After introduction of the vector, cells may be grown in rich medium for about 1-2 days, after which the medium may be switched to selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows the growth and recovery of cells that successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate for the cell type. Transient production, for example by transient transfection or infection, may be used as well. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0223] Any number of selection systems may be used to recover transformed or transduced cell lines, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)) genes, which can be used in tk- or aprt- cells, respectively. Similarly, antimetabolite, antibiotic, or herbicide resistance; for example, dhfr, which confers resistance to methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); and als or pat, which confer resistance to chlorsulfuron and phosphinothricin acetyltransferase, respectively (Murry, supra), can be used as the basis for selection. Additional selectable genes have been described, such as trpB, which allows cells to utilize indole instead of tryptophan, or hisD, which allows cells to utilize histinol instead of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047-51 (1988)). The use of visible markers has become widespread, and markers such as green fluorescent protein (GFP) and other fluorescent proteins (e.g., RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, and luciferase and its substrate luciferin are widely used not only to identify transformants, but also to quantify the amount of transient or stable protein expression resulting from specific vector systems (see, for example, Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0224] Also included are high-throughput protein production systems or micro-production systems.Certain embodiments may utilize hexahistidine fusion tags for protein expression and purification, for example, on metal chelate modified slide surfaces or MagneHis Ni particles (see, for example, Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., Methods Mol Biol. 498:129-41, 2009).Also included are high-throughput cell-free protein expression systems (see, for example, Sitaraman et al., Methods Mol Biol. 498:229-44, 2009).

[0225] A variety of protocols are known in the art for detecting and measuring the expression of polynucleotide-encoded products using binding agents or antibodies, such as polyclonal or monoclonal antibodies specific for the product. Examples include enzyme-linked immunosorbent assay (ELISA), Western immunoblot, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other assays are described, among others, in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

[0226] A wide variety of labeling and conjugation techniques are known to those skilled in the art and can be used in various nucleic acid and amino acid assays. Means of producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides include oligo-labeling, nick-translation, end-labeling or PCR amplification using labeled nucleotides. Alternatively, the sequence or any part thereof may be cloned into a vector for the production of mRNA probes. Such vectors are known in the art and commercially available and may be used to synthesize RNA probes in vitro by adding a suitable RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be carried out using a variety of commercially available kits. Suitable reporter molecules or labels that may be used include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the like.

[0227] The host cell transformed with one or more polynucleotide sequences of interest can be cultured under suitable conditions for protein expression and recovery from cell culture.Certain specific embodiments utilize serum-free cell expression systems.Examples include HEK293 cells and CHO cells that can grow in serum-free medium (see, for example, Rosser et al., Protein Expr. Purif. 40:237-43, 2005; and U.S. Patent No. 6,210,922).

[0228] The activatable proprotein produced by a recombinant cell may be secreted or contained intracellularly depending on the sequence and / or vector used. As will be understood by those skilled in the art, the expression vector containing the polynucleotide may be designed to contain a signal sequence that directs the secretion of the encoded polypeptide through a prokaryotic or eukaryotic cell membrane. Other recombinant constructs may be used to link the sequence encoding the polypeptide of interest to a nucleotide sequence encoding a polypeptide domain that facilitates the purification and / or detection of soluble proteins. Examples of such domains include cleavable and non-cleavable affinity purification and epitope tags, such as avidin, FLAG tags, polyhistidine tags (e.g., 6xHis), cMyc tags, V5 tags, glutathione S-transferase (GST) tags, and the like.

[0229] Proteins produced by recombinant cells can be purified and characterized according to a variety of techniques known to those of skill in the art. Exemplary systems for performing protein purification and for analyzing protein purity include fast protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems), high performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradients, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reverse phase, ceramic HYPERD® ion exchange chromatography, and hydrophobic interaction columns (HIC), among others known in the art. Also included are analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblots, Bradford, and ELISA, which can typically be utilized during any step of the production or purification process to measure the purity of a protein composition.

[0230] Also included are methods for concentrating activatable proproteins and compositions comprising concentrated, soluble, activatable proproteins. In some embodiments, such concentrated solutions of at least one activatable proprotein are at least about or at least about 100% soluble. Also contains protein at a concentration of about 5 mg / mL, about or at least about 8 mg / mL, about or at least about 10 mg / mL, about or at least about 15 mg / mL, about or at least about 20 mg / mL, or higher.

[0231] In some aspects, such compositions can be substantially monodisperse, meaning that the activatable proprotein is present predominantly (i.e., at least about 90% or more) in one apparent molecular weight form, as assessed, for example, by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0232] In some aspects, such compositions have a purity of at least about 90% (based on protein), or in some aspects at least about 95% purity, or in some embodiments at least 98% purity. Purity can be determined via any routine analytical method known in the art.

[0233] In some aspects, such compositions have a high molecular weight aggregate content of less than about 10% relative to the total amount of protein present, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 5%, or in some aspects, such compositions have a high molecular weight aggregate content of less than about 3%, or in some embodiments, such compositions have a high molecular weight aggregate content of less than about 1%. High molecular weight aggregate content can be determined via a variety of analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0234] Examples of concentration approaches contemplated herein include lyophilization, which is typically used when the solution contains few soluble components other than the protein of interest. Lyophilization is often performed after an HPLC run and can remove most or all volatile components from the mixture. Also included are ultracentrifugation techniques, which typically use one or more selectively permeable membranes to concentrate protein solutions. The membrane allows water and small molecules to pass through and retains the protein; the solution can be forced against the membrane by mechanical pumps, gas pressure, or centrifugation, among other techniques.

[0235] In certain embodiments, the activatable proprotein in the composition has a purity of at least about 90% as measured according to routine techniques in the art. In certain embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the activatable proprotein composition has a purity of at least about 95%, or at least about 97%, or at least 98%, or at least 99%. In some embodiments, such as when used as a reference or research reagent, the activatable proprotein may be a lower purity activatable proprotein and may have a purity of at least about 50%, at least 60%, at least 70%, or at least 80%. Purity can be measured as a whole or in relation to a selected component, such as other proteins, and may be, for example, a protein-based purity.

[0236] The purified activatable proprotein can also be characterized according to its biological characteristics. Binding affinity and binding kinetics can be measured according to various techniques known in the art, such as Biacore® and related techniques that utilize surface plasmon resonance (SPR), an optical phenomenon that allows real-time detection of unlabeled interactants. SPR-based biosensors can be used in active enrichment, screening, and characterization determinations for both affinity and kinetics. The presence or level of one or more biological activities can be measured according to cell-based assays, including assays that utilize at least one IL-2 receptor, which is optionally functionally linked to a readout or indicator, such as a fluorescent or luminescent indicator of biological activity, as described herein.

[0237] In certain embodiments, as described above, the activatable proprotein composition is substantially endotoxin-free, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, more preferably about 99.99% endotoxin-free. The presence of endotoxin can be detected according to routine techniques in the art, as described herein. In specific embodiments, the activatable proprotein composition is made from eukaryotic cells, such as mammalian or human cells, in a substantially serum-free medium. In certain embodiments, as described herein, the activatable proprotein composition has an endotoxin content of less than about 10 EU / mg activatable proprotein, or less than about 5 EU / mg activatable proprotein, less than about 3 EU / mg activatable proprotein, or less than about 1 EU / mg activatable proprotein.

[0238] In certain embodiments, the activatable proprotein composition comprises less than about 10 wt / wt% high molecular weight aggregates, or less than about 5 wt / wt% high molecular weight aggregates, or less than about 2 wt / wt% high molecular weight aggregates, or less than about or less than about 1% wt / wt high molecular weight aggregates.

[0239] Also included are protein-based analytical assays and methods that can be used to assess, for example, protein purity, size, solubility, and degree of aggregation, among other characteristics. Protein purity can be assessed in a number of ways. For example, purity can be assessed based on primary structure, higher order structure, size, charge, hydrophobicity, and glycosylation. Examples of methods for assessing primary structure include N- and C-terminal sequencing and peptide mapping (see, for example, Allen et al., Biologicals. 24:255-275, 1996). Exemplary methods for assessing conformation include circular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, e.g., Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity with conformation-sensitive antibodies. Conformation can also be assessed as a function of various parameters, such as pH, temperature, or added salt. Exemplary methods for assessing protein characteristics, such as size, include analytical ultracentrifugation and size-exclusion HPLC (SEC-HPLC), and exemplary methods for measuring charge include ion-exchange chromatography and isoelectric focusing. Hydrophobicity can be assessed, for example, by reversed-phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (eg, clearance), conformation or stability, receptor binding, and protein function and can be assessed, for example, by mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0240] As mentioned above, certain embodiments include the use of SEC-HPLC to evaluate protein characteristics such as purity, size (e.g., size uniformity) or degree of aggregation, and / or to purify proteins, among other uses. SEC, which also includes gel filtration chromatography (GFC) and gel permeation chromatography (GPC), refers to a chromatographic method in which molecules in solution are separated in a porous material based on their size, or more specifically their hydrodynamic volume, diffusion coefficient, and / or surface properties. The process is generally used to separate biological molecules and to determine the molecular weight and molecular weight distribution of polymers. Typically, a biological or protein sample (e.g., a protein extract produced according to the protein expression methods provided herein and known in the art) is loaded into a selected size-exclusion column with a defined stationary phase (porous material), preferably a phase that does not interact with the proteins in the sample. In certain aspects, the stationary phase is composed of inert particles packed into a dense three-dimensional matrix in a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. Stationary phase particles typically have small pores and / or channels into which only molecules smaller than a certain size can enter. Larger particles are therefore excluded from these pores and channels, and due to their limited interaction with the stationary phase, they elute as a "totally excluded" peak at the beginning of the experiment. Smaller molecules that can fit into the pores are removed from the flowing mobile phase, and the time they remain immobilized in the pores of the stationary phase depends in part on the distance to the pores they penetrate. Their removal from the mobile phase stream means that they take longer to elute from the column, resulting in separation between particles based on their size differences. A given size exclusion column has a molecular weight range that it can separate. Overall, molecules larger than the upper limit are not captured by the stationary phase, molecules smaller than the lower limit enter the solid phase completely and elute as a single band, and molecules within the range elute at different rates defined by their properties, such as hydrodynamic volume.For examples of these methods in pharmaceutical protein practice, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

[0241] Protein purity for clinical applications has also been discussed, for example, by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, but are not limited to, the LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids, which provides high-throughput analysis of protein titer, sizing, and purity analysis. In certain non-limiting embodiments, clinical grade activatable proproteins can be obtained by utilizing a combination of chromatographic materials in at least two orthogonal steps, among other methods (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, Eds., Smales and James, Humana Press Inc., 2005). Typically, the protein agent (e.g., activatable proprotein) is substantially endotoxin-free when measured according to techniques known in the art and described herein.

[0242] Protein solubility assays are also included. Such assays can be utilized, for example, to determine optimal growth and purification conditions for recombinant production, to optimize the selection of buffer(s), and to optimize the selection of activatable proproteins and their variants. Solubility or aggregation can be evaluated according to a variety of parameters, including temperature, pH, salts, and the presence or absence of other additives. Examples of solubility screening assays include, but are not limited to, microplate-based methods that measure protein solubility using turbidity or other measures as an endpoint, high-throughput assays for analysis of purified recombinant protein solubility (see, e.g., Stenvall et al., Biochim Biophys Acta. 1752:6-10, 2005), assays that use structural complementation of genetic marker proteins to monitor and measure protein folding and solubility in vivo (see, e.g., Wigley et al., Nature Biotechnology. 19:131-136, 2001), and electrochemical screening of recombinant protein solubility in Escherichia coli using scanning electrochemical microscopy (SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering. 96:1008-1013, 2006), among others. Activatable proproteins with increased solubility (or reduced aggregation) can be identified or selected according to routine techniques in the art, including simple in vivo assays for protein solubility (see, e.g., Maxwell et al., Protein Sci. 8:1908-11, 1999).

[0243] Protein solubility and aggregation can also be measured by dynamic light scattering techniques. Aggregation is a general term that encompasses several types of interactions or characteristics, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregates is typically considered undesirable due to concerns that aggregates may cause immunogenic responses (e.g., small aggregates) or cause adverse events upon administration (e.g., particulates). Dynamic light scattering refers to a technique that can be used to determine the size distribution profile of small particles in suspension or polymers such as proteins in solution. This technique, also known as photon correlation spectroscopy (PCS) or quasi-elastic light scattering (QELS), uses scattered light to measure the diffusion rate of protein particles. Variations in the scattering intensity due to Brownian motion of molecules and particles in solution can be observed. This motion data can be conventionally processed to derive a size distribution of the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particle. The hydrodynamic size depends on both mass and shape (conformation). Dynamic scattering can detect the presence of very small amounts (<0.01% by weight) of aggregated protein, even in samples containing a large mass range. Similarly, it can be used to compare the stability of different formulations, including applications that rely on real-time monitoring of changes at elevated temperatures, for example. Thus, certain embodiments include the use of dynamic light scattering to analyze the solubility and / or presence of aggregates in samples containing the activatable proproteins of the present disclosure.

[0244] Although the foregoing embodiments have been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of this disclosure that certain changes and modifications can be made to the present disclosure without departing from the spirit or scope of the appended claims. The following examples are provided for illustration only, and not for limitation. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0245] Example 1 Engineering "IL-2-linker-IL-2Rα" and "IL-2Rα-linker-IL-2" activatable proproteins To reduce the toxicity of IL-2-related therapeutics, IL-2-linker-IL-2Rα fusion proteins (herein referred to as ILR fusion proteins) and IL-2Rα-linker-IL-2 fusion proteins (herein referred to as RLI fusion proteins) were generated as prodrugs, or activatable proproteins. The prodrugs have very low activity in their activated form. Full or near-full activity can be restored by protease cleavage of the protease-specific linker sequence designed into the prodrug (see, e.g., Figures 4A-4E).

[0246] An exemplary fusion protein was used, human IL-2-T3 with a triple mutation of V69A, Q74P, and I128T, which has higher binding affinity to IL-2Rα. A TEV protease cleavage site was used to restore IL-2 activity, providing proof of concept.

[0247] Plasmids encoding single chain IL-2-linker-IL-2Rα (ILR) and IL-2Rα-linker-IL-2 (RLI) with or without Fc fusion were constructed by standard gene synthesis and subsequently subcloned into pTT5 expression vector. Schematic diagrams of exemplary ILR fusion protein formats are shown in Figures 2A, 2B, 2D, 2E, 2G, and 2H. Schematic diagrams of exemplary RLI fusion protein formats are shown in Figures 3A, 3B, 3D, 3E, 3G, and 3H.

[0248] Exemplary proteins for the IL-2-stable linker-IL-2Rα format with a C-terminal His tag (FIG. 2A) include P1522 and P1525. Exemplary proteins for the IL-2-TEV-IL-2Rα format with a C-terminal His tag (FIG. 2B) include P1630, P1664, and P1667. Exemplary proteins for the Fc-TEV-IL-2-stable linker-IL-2Rα format (FIG. 2D) include P1523 and P1526. Exemplary proteins for the Fc-stable linker-IL-2-TEV-IL-2Rα format (FIG. 2E) include P1631, P1665, and P1668. Exemplary proteins for the IL-2-stable linker-IL-2Rα-TEV-Fc format (FIG. 2G) include P1524 and P1527. Exemplary proteins in the IL-2-TEV-IL-2Rα-stable linker-Fc format (FIG. 2H) include P1632, P1666, and P1669.

[0249] Exemplary proteins for the IL-2Rα-stable linker-IL-2 format with a C-terminal His tag (FIG. 3A) include P1528 and P1531. Exemplary proteins for the IL-2Rα-TEV-IL-2 format with a C-terminal His tag (FIG. 3B) include P1633, P1773, and P1776. Exemplary proteins for the Fc-TEV-IL-2Rα-stable linker-IL-2 format (FIG. 3D) include P1529 and P1532. Exemplary proteins for the Fc-stable linker-IL-2Rα-TEV-IL-2 format (FIG. 3E) include P1634, P1774, and P1777. Exemplary proteins for the IL-2Rα-stable linker-IL-2-TEV-Fc format (FIG. 3G) include P1530 ​​and P1533. Exemplary proteins in the IL-2Rα-TEV-IL-2-stable linker-Fc format (Figure 3H) include P1635, P1775, and P1778.

[0250] In P1522, P1523, P1524, P1528, P1529, and P1530, the linker length is 5 amino acids between IL-2 and IL-2Rα. In P1525, P1526, P1527, P1531, P1532, and P1533, the linker length is 10 amino acids between IL-2 and IL-2Rα. In P1630, P1631, P1632, P1633, P1634, and P1635, the linker length is 11 amino acids between IL-2 and IL-2Rα. In P1664, P1665, P1666, P1773, P1774, and P1775, the linker length is 15 amino acids between IL-2 and IL-2Rα. In P1667, P1668, P1669, P1776, P1777, and P1778, the linker length is 19 amino acids between IL-2 and IL-2Rα.

[0251] For the ILR format, a real protease cleavage site (PS) was introduced into the linker between IL-2 and IL-2Rα. A potential O-glycosylation site in IL-2 was substituted with alanine (T3A). A disulfide bond was introduced between IL-2 and IL-2Rα by introducing E61C into IL-2 and K38C into IL-2Rα. Exemplary proteins include P1719 (IL-2-PS-IL-2Rα-His6), P1720 (Fc-stable linker-IL-2-PS-IL-2Rα), P1721 (IL-2-PS-IL-2Rα-stable linker-Fc), P1722 (IL-2-PS-IL-2Rα-His6), P1723 (Fc-stable linker-IL-2-PS-IL-2Rα), and P1724 (IL-2-PS-IL-2Rα-stable linker-Fc).

[0252] For the Fc-stable linker-IL-2Rα-PS-IL-2 format, a real protease cleavage site (PS) was introduced into the linker between IL-2Rα and IL-2. A potential O-glycosylation site in IL-2 was replaced with alanine (T3A). A disulfide bond was introduced between IL-2 and IL-2Rα. At least one cysteine ​​mutation was introduced into K35, R38 or E61 of IL-2 and into D04, H120, K38 or S39 of IL-2Rα. Exemplary proteins include P1725, P1726, P1727, P1728, P1729 and P1730.

[0253] For activatable proprotein design, a real protease cleavage site (PS) was introduced into the linker between IL-2 and IL-2Rα for the IL-2-PS-IL-2Rα-stable linker-Fc format. A potential O-glycosylation site in IL-2 was replaced with alanine (T3A). A plasmid encoding IL-2-PS-IL-2Rα-stable linker-Fc was constructed by standard gene synthesis and subcloned into a pTT5 expression vector with the linker flanked by protease cleavage sites. Exemplary proteins include P1779, P1780, P1781, P1782, P1783, P1784, and P1785. P1786 was generated as a control protein with no cleavage site between IL-2 and IL-2Rα.

[0254] For the activatable proprotein design, different actual protease cleavage sites (PS) were introduced into the linker between Fc / IL-2 and IL-2 / IL-2Rα, respectively. A potential O-glycosylation site in IL-2 was replaced with alanine (T3A). A potential N-glycosylation site in IL-2R was replaced with alanine in one construct (N49A and N68A). IL-2-D10 was also tested in one construct. Plasmids encoding Fc-PS1-IL-2-PS2-IL2Rα were constructed by standard gene synthesis and subcloned into the pTT5 expression vector with linkers flanked by protease cleavage sites. Exemplary proteins include P1834, P1835, P1836, P1837, P1838, P1839, P1840, P1841, P1842, P1843, P1844, P1845, P1846, P1847, P1848, P1849, and P1850.

[0255] IL-2 muteins with lower binding affinity to wild-type IL-2 and IL-2Rα were also tested in the Fc-stable linker-IL-2-TEV-IL-2Rα format. Potential O-glycosylation sites were replaced with alanine (T3A). IL-2 muteins tested include IL-2-F42A, IL-2-Y45A, and IL-2-F42A-Y45A. Exemplary proteins include P1946, P1947, P1948, and P1949. The combination of IL-2-E61S / IL-2Rα-K38S was also tested. Exemplary proteins include P1972.

[0256] The ILR format was also tested in an antibody fusion format. The ILR was fused to the C-terminus of the antibody heavy chain using a protease cleavage site between the heavy chain and IL-2 or between IL-2 and IL-2Rα. The potential O-glycosylation site in IL-2 was replaced with alanine (T3A) and the C-terminal lysine (K) on the heavy chain was deleted. Since IgG4-Fd was used, cysteine ​​217 on the heavy chain was replaced with serine. Exemplary proteins include P14501950, P14501951, P14501952, and P14501953.

[0257] Production, purification and characterization. Fc fusion proteins were produced by transient transfection in Expi293 cells and purified by a two-step purification process including MabSelect SuRe chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare). His-tagged proteins were produced by transient transfection in Expi293 cells and purified by a two-step purification process including nickel affinity chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare).

[0258] The purified proteins were characterized by SDS-PAGE to assess purity, which showed good purity, for example, as shown in Figures 6A, 6B, 10A, 10B, 13A, 13B, 16A, 16B, 19A, 19B, 22A, 22B, 25A, 25B, 27A, and 27B.

[0259] Protease cleavage was performed on purified proteins with corresponding cleavage sites. The proteases tested were: TEV, uPA (R&D, Cat. No. 1310-SE-010), matriptase (R&D, Cat. No. 3946-SEB-010) and MMP-2 (R&D, Cat. No. 902-MP-010). As shown in Figure 6C, P1529, P1532, P1630, P1631 and P1632 could not be cleaved by TEV, but other proteins could be cleaved by TEV. As shown in Figure 10C, P1664, P1665, P1666, P1667, P1668 and P1669 could be cleaved by TEV. As shown in Figure 13C, P1719, P1721, P1722, P1723, P1724, P1725 and P1726 could be partially cleaved by uPA protease. As shown in Figure 16C, P1773, P1774, P1775, P1776, P1777 and P1778 could be cleaved by TEV, and P1779, P1780, P1781, P1782, P1783, P1784 and P1785 could be cleaved by uPA protease.

[0260] As shown in Figure 19C, the purified proteins could be partially or completely cleaved by uPA, matriptase or MMP-2. As shown in Figure 19D, P1842 and P1847 could be simultaneously cleaved by uPA and MMP-2. As shown in Figure 22C, P1946, P1947, P1948 and P1949 could be partially cleaved by TEV. As shown in Figure 25C, P14501950, P14501951, P14501952 and P14501953 could be completely or partially cleaved by MMP-2, uPA or matriptase. As shown in Figure 27C, P1972 could be partially cleaved by TEV.

[0261] The purified proteins were also characterized by high performance liquid chromatography (HPLC) for assessment of homogeneity. HPLC analysis was performed using a Nanofilm SEC-250 column (Sepax) and an Agilent 1260 according to the manufacturer's instructions. Representative HPLC results are shown in Figures 7A-7J, 11A-11F, 14A-14D, 17A-17D, 20A-20D, 23A-23D, 26A-26D, and 27D. The majority of the proteins showed one single peak indicating good homogeneity.

[0262] Functional assays - proliferation. Proliferation assays were performed on purified proteins before and after cleavage. M-07e cells (IL-2Rβ / γc) were cultured in RPMI1640 supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, Mo7e cells were harvested in their logarithmic growth phase and washed twice with PBS. Cell suspensions (2×10 cells) were cultured in RPMI1640 supplemented with 20% fetal bovine serum (FBS), 1% non-essential amino acids (NEAA), and 10% 5637 cell culture supernatant. To measure cytokine-dependent cell proliferation, Mo7e cells were harvested in their logarithmic growth phase and washed twice with PBS. 4 90 μl of IL-2 / well were seeded into a 96-well plate and incubated for 4 h at 37°C and 5% CO2 in assay medium (RPMI1640 supplemented with 10% FBS and 1% NEAA) for cytokine depletion. IL-2 control and purified protein samples used in the assay were prepared in assay medium at an initial concentration of 300 nM, followed by 1 / 3 serial dilutions. 10 μl of diluted protein was added to the corresponding wells and incubated at 37°C and 5% CO2 for 72 h. A colorimetric assay using Cell Counting Kit-8 (CCK-8, Dojindo, CK04) was performed to measure the amount of viable cells. The results are shown in Figures 8A-8L, 9A-9E, 12A-12F, 15A-15E, 18A-18N, 21A-21Q, 24A-24D, and 28.

[0263] No activity was detected for fusion proteins lacking the TEV cleavage site from the IL-2-stable linker-IL-2Rα formats (P1522 and P1525) and IL-2Rα-stable linker-IL-2 formats (P1528 and P1531).

[0264] No activity was detected for the fusion proteins before TEV cleavage from the Fc-TEV-IL-2-stable linker-IL-2Rα formats (P1523 and P1526), ​​IL-2-stable linker-IL-2Rα-TEV-Fc formats (P1524 and P1527), Fc-TEV-IL-2Rα-stable linker-IL-2 formats (P1529 and P1532) and IL-2Rα-stable linker-IL-2-TEV-Fc formats (P1530 ​​and P1533). For these fusion proteins, IL-2 activity was not restored after cleavage with TEV.

[0265] For formats with a TEV cleavage site between IL-2 and IL-2Rα, P1630, P1631, and P1632 showed no activity before cleavage with TEV and could not be cleaved by TEV; P1635 showed no activity before or after cleavage with TEV; and P1633 and P1634 showed low activity before cleavage with TEV and regained full or partial activity after cleavage with TEV.

[0266] For fusion proteins with a longer cleavable linker (TEV cleavage site) between IL-2 and IL-2Rα, P1664, P1665, P1666, P1667, P1668, P1669, P1773, P1774, P1776, and P1777 showed very low activity before cleavage with TEV and regained full or partial activity after cleavage with TEV, whereas P1775 and P1778 showed no or very low activity before cleavage with TEV and could not regain activity after cleavage with TEV.

[0267] For fusion proteins with an actual protease cleavage site in the linker between IL-2 and IL-2Rα, P1779, P1780, P1781, P1782, P1783, and P1785 showed no or very low activity before protease cleavage and activity was restored after protease cleavage, while P1786, as a negative control, showed no activity before or after protease cleavage.

[0268] For the ILR format with a disulfide bond between IL-2 and IL-2Rα, P1719 P1721, P1722, P1723, and P1724 showed no or low activity before protease cleavage and partial or full recovery of activity after protease cleavage.

[0269] For the Fc-PS1-IL-2-PS2-IL2Rα format, as shown in Figures 21A-21O, the fusion proteins showed no or very low activity before protease cleavage, and activity was partially restored after protease cleavage at PS2. For P1842 and P1847, different cleavage combinations were tested: single cleavage at PS1, single cleavage at PS2, and double cleavage at both PS1 and PS2. For P1842, low activity was restored after single cleavage at PS1 or PS2, and full activity was restored after double cleavage at both PS1 and PS2. For P1847 with superkine D10, low activity was detected before protease cleavage, and full activity was restored after single cleavage at PS1 or PS2 and double cleavage at both PS1 and PS2, and indeed after double cleavage this construct showed higher activity than wild type IL-2.

[0270] For fusion proteins with wild-type IL-2 or IL-2 muteins with lower binding affinity to IL-2Rα, P1946, P1947, P1948, and P1949 showed low activity before protease cleavage and regained full activity after cleavage. P1947, P1948, and P1949 with IL-2 muteins showed higher activity than P1946 before protease cleavage.

[0271] P1972 carrying IL-2-E61S and IL-2Rα-K38S showed low activity before cleavage with TEV and full activity was restored after cleavage. The present invention provides, for example, the following items. (Item 1) An activatable proprotein homodimer comprising a first polypeptide and a second polypeptide, (a) the first polypeptide and the second polypeptide comprise, in N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein; or (b) the first polypeptide and the second polypeptide comprise, in an N-to-C-terminal or C-to-N-terminal direction, a binding moiety, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein; said binding moiety of said first polypeptide binds to said binding moiety of said second polypeptide, said IL-2 protein of said first polypeptide binds to said IL-2 binding protein of said second polypeptide, said IL-2 binding protein of said first polypeptide binds to said IL-2 protein of said second polypeptide, said binding masking a binding site of IL-2 protein(s) that would otherwise bind to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell in vitro or in vivo, and at least one of said first or said second linker is a cleavable linker; or (c) the first and second polypeptides comprise, in an N-to-C-terminal or C-to-N-terminal direction, an IL-2 protein, a first linker, an IL-2 binding protein, a second linker, and an affinity purification tag; or (d) the first and second polypeptides comprise, in an N-to-C-terminal or C-to-N-terminal direction, an IL-2 binding protein, a first linker, an IL-2 protein, a second linker, and an affinity purification tag; An activatable proprotein homodimer, wherein the IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and the IL-2 binding protein of the first polypeptide binds to the IL-2 protein of the second polypeptide, said binding masking a binding site of IL-2 protein(s) that would otherwise bind to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of an immune cell in vitro or in vivo, and wherein the first linker is a cleavable linker. (Item 2) 2. The activatable proprotein homodimer of item 1, wherein the first and second IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence selected from Table S1, optionally an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 21 to 153 of SEQ ID NO:1 (full length wild-type human IL-2), and optionally comprising a C145X (wherein X is any amino acid) or C145S substitution as defined by SEQ ID NO:1. (Item 3) 3. The activatable proprotein homodimer of item 1 or 2, wherein the first and second IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:2 (mature human IL-2 with a C125S substitution), optionally wherein the IL-2 protein retains the S125 residue defined by SEQ ID NO:2. (Item 4) 4. The activatable proprotein gene of any one of items 1 to 3, wherein the first and second IL-2 proteins comprise one or more substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined by SEQ ID NO:2. Monodimer. (Item 5) 5. The activatable proprotein homodimer of claim 4, wherein the first IL-2 protein forms a disulfide bond with the second IL-2 binding protein and the second IL-2 protein forms a disulfide bond with the first IL-2 binding protein, optionally via one or more of the cysteines described in claim 4 and one or more cysteines in the first and second IL-2 binding protein(s). (Item 6) 6. The activatable proprotein of any one of items 1 to 5, wherein said first and second IL-2 proteins comprise one or more amino acid substitutions at positions 69, 74, and / or 128 as defined by SEQ ID NO:2, optionally wherein said one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined by SEQ ID NO:2. (Item 7) and wherein the first and second IL-2 proteins comprise one or more amino acid substitutions at positions T3, R38, F42, Y45, E61, E62, E68, and / or L72 as defined by SEQ ID NO:2, optionally the one or more amino acid substitutions include combinations thereof: T3A; R38A and R38K; F42A, F42G, F42S, F42T, F42Q, F42E, F42N, F42D, F42R, F42K, and F42I; Y45A, Y45G, Y45S, Y45T, Y45Q, Y45E, Y45N, Y45D, Y45R, and Y45K; E61S; E62A and E62L; E68A and E68V; and 7. The activatable proprotein homodimer of any one of items 1 to 6, selected from a combination selected from: L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R, and L72K, optionally F42A, Y45A, and L72G; R38K, F42Q, Y45N, E62L, and E68V; R38K, F42Q, Y45E, and E68V; R38A, F42I, Y45N, E62L, and E68V; R38K, F42K, Y45R, E62L, and E68V; R38K, F42I, Y45E, and E68V; and R38A, F42A, Y45A, and E62A. (Item 8) 8. The activatable proprotein homodimer of any one of items 1 to 7, wherein said first and second IL-2 proteins comprise, consist of, or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to SEQ ID NO:3 (mature human IL-2 "D10" variant), optionally wherein said IL-2 protein retains any one or more of the Q74H, L80F, R81D, L85V, I86V, and / or I92F substitutions defined by SEQ ID NO:3. (Item 9) 9. The activatable proprotein homodimer of any one of items 1 to 8, wherein said first and second IL-2 binding proteins comprise a first and second IL-2Rα protein, or a first and second antibody or antigen-binding fragment thereof that specifically binds to said IL-2 protein(s), optionally a bispecific antibody or antigen-binding fragment thereof. (Item 10) 10. The activatable proprotein homodimer of item 9, wherein the first and second IL-2Rα proteins comprise, consist of, or consist essentially of an amino acid sequence selected from Table S2, optionally an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to amino acids 22 to 187 of SEQ ID NO: 4 (full-length wild-type human IL-2Rα). (Item 11) 11. The activatable proprotein homodimer according to item 9 or 10, wherein the first and second IL-2Rα proteins comprise one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C, and / or a K38S substitution as defined by SEQ ID NO:6 (human IL-2Rα Sushi1-Sushi2 domain). (Item 12) wherein the first IL-2Rα protein forms a disulfide bond with the second IL-2 protein, and the second IL-2Rα protein forms a disulfide bond with the first IL-2 protein, optionally via one or more of the cysteines according to item 11 and one or more cysteines in the IL-2 protein, optionally one or more of the cysteines according to item 4, optionally one or more cysteine ​​pairs selected from IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, IL2-R38C and IL2Rα-H120C, IL2-T41C and IL2Rα-I118C, IL2-F42C and IL2Rα-N27C, IL2-E61C and IL2Rα-K38C, IL2-E61C and IL2Rα-S39C, and IL2-V69C and IL2Rα-L42C, The disulfide bond between the IL-2 protein and the IL-2Rα protein is reg 12. The activatable proprotein homodimer according to any one of items 9 to 11, which masks the binding site of the IL-2 protein that preferentially binds to the IL-2Rα / β / γc chain expressed on (Item 13) 13. The activatable proprotein homodimer of any one of items 9 to 12, wherein the first and second IL-2Rα proteins comprise an alanine substitution at positions 49 and / or 68 as defined by SEQ ID NO:6. (Item 14) 10. The activatable proprotein homodimer of item 9, wherein the first and second antibodies or antigen-binding fragments thereof that specifically bind to the IL-2 protein are selected from one or more of a whole antibody, a Fab, a Fab', a F(ab')2, a monospecific Fab2, a bispecific Fab2, a FV, a single-chain Fv (scFv), a scFV-Fc, a nanobody, a diabody, a camelid antibody, and a minibody, and optionally the antibody is NARA1 or an antigen-binding fragment thereof. (Item 15) 15. The activatable proprotein homodimer of any one of items 1 to 14, wherein the binding moieties of (a) and / or (b) do not bind to the IL-2 protein or to the IL-2 binding protein. (Item 16) 15. The activatable proprotein homodimer according to any one of items 1 to 14, wherein the binding moieties of (a) and / or (b) bind to the IL-2 protein. (Item 17) 17. The activatable proprotein homodimer according to any one of items 1 to 16, wherein the binding portions of the first and second polypeptides of (a) and / or (b) bind to each other via at least one non-covalent interaction, optionally forming a homodimer. (Item 18) 18. The activatable proprotein homodimer according to any one of items 1 to 17, wherein the binding moieties of the first and second polypeptides of (a) and / or (b) are linked to each other via at least one covalent bond, optionally forming a homodimer. (Item 19) the at least one covalent bond comprises at least one disulfide bond. 19. An activatable proprotein homodimer according to 18. (Item 20) 20. The activatable proprotein homodimer according to any one of items 1 to 19, wherein the binding moieties of the first and second polypeptides of (a) and / or (b) are selected from Table M1. (Item 21) 21. The activatable proprotein homodimer of any one of items 1 to 20, wherein the binding portions of the first and second polypeptides of (a) or (b) comprise an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof. (Item 22) 22. The activatable proprotein of any one of items 1 to 21, wherein the binding portions of the first and second polypeptides of (a) and / or (b) comprise the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or CL domains of an immunoglobulin, including fragments and variants thereof. (Item 23) 23. The activatable proprotein homodimer of item 21 or 22, wherein the binding portions of the first and second polypeptides of (a) and / or (b) comprise, in an N-to-C-terminal direction: (1) an antigen-binding domain of an immunoglobulin, including antigen-binding fragments and variants thereof; and (2) the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or CL domains of an immunoglobulin, including fragments and variants thereof. (Item 24) 24. The activatable proprotein homodimer of any one of items 21 to 23, wherein the antigen-binding domain comprises an immunoglobulin VH or VL domain, including antigen-binding fragments and variants thereof. (Item 25) 25. The activatable proprotein homodimer according to any one of items 1 to 24, wherein the binding portions of the first and second polypeptides of (a) and / or (b) do not bind to an antigen. (Item 26) 26. The activatable proprotein homodimer of any one of items 1 to 25, wherein the binding portions of the first and second polypeptides of (a) and / or (b) comprise an immunoglobulin CH2CH3 domain. (Item 27) 27. The activatable proprotein homodimer of any one of items 21 to 26, wherein the immunoglobulin is from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. (Item 29) 29. The activatable proprotein homodimer of any one of the preceding claims, wherein the binding portions of the first and second polypeptides of (a) and / or (b) comprise leucine zipper peptides. (Item 30) 30. The activatable proprotein homodimer of any one of items 1 to 29, wherein said affinity purification tag of (c) and / or (d) is selected from a polyhistidine tag (optionally a hexahistidine tag), a VSV-G tag, a universal tag, a Strep tag, a S tag, a S1 tag, a Phe tag, a Cys tag, an Asp tag, an Arg tag, a Myc epitope tag, a KT3 epitope tag, an HSV epitope tag, a histidine affinity tag, a hemagglutinin (HA) tag, a FLAG epitope tag, an E2 epitope tag, a V5 tag, a T7 tag, an AU5 epitope tag, and an AU1 epitope tag. (Item 31) 31. The activatable proprotein homodimer according to any one of the preceding items, wherein the cleavable linker comprises a protease cleavage site, optionally wherein the cleavable linker is selected from Table S3. (Item 32) 32. The activatable proprotein homodimer of claim 31, wherein the protease cleavage site is cleavable by a protease selected from one or more of a metalloprotease, a serine protease, a cysteine ​​protease, and an aspartic acid protease. (Item 33) 33. The activatable proprotein homodimer of item 31 or 32, wherein the protease cleavage site is cleavable by a protease selected from one or more of MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, matriptase, uPA, FAP, legumain, PSA, kallikrein, cathepsin A, and cathepsin B. (Item 34) The first linker and / or the second linker are about 1 to 50, about 1 to 40, about 1 to 30, about 1 to 20, about 1 to 10, about 1 to 5, about 1 to 4, about 1 to 3 amino acids in length, or about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, 35. The activatable proprotein homodimer of any one of claims 1 to 33, which is about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50 amino acids in length. 35. The activatable proprotein homodimer of any one of items 1 to 34, wherein the first linker of (a) and / or (b) is a cleavable linker and the second linker of (a) and / or (b) is a non-cleavable linker. (Item 36) 36. The activatable proprotein homodimer of item 35, wherein cleavage, optionally by a protease, of the first linker of (a) and / or (b) exposes the binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of the immune cell in vitro or in vivo. (Item 37) 35. The activatable proprotein homodimer of any one of the preceding items, wherein the first linker of (a) and / or (b) is a non-cleavable linker and the second linker of (a) and / or (b) is a cleavable linker. (Item 38) 38. The activatable proprotein homodimer of item 37, wherein cleavage, optionally by a protease, of the second linker of (a) and / or (b) exposes the binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of the immune cell in vitro or in vivo. (Item 39) 35. The method according to any one of items 1 to 34, wherein cleavage, optionally by a protease, of the first linker of (c) and / or (d) exposes the binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of the immune cell in vitro or in vivo. Activatable proprotein homodimer. (Item 40) 40. The activatable proprotein homodimer of any one of the preceding items, wherein the immune cell is selected from one or more of a T cell, a B cell, a natural killer cell, a monocyte, and a macrophage. (Item 41) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (a) comprise, in N-to-C-terminal direction, the binding moiety, the first linker, the IL-2 protein, the second linker, and the IL-2 binding protein. (Item 42) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (a) comprise, in an N-to-C-terminal direction, the IL-2 binding protein, the first linker, the IL-2 protein, the second linker, and the binding moiety. (Item 43) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (b) comprise, in N-to-C-terminal direction, the binding moiety, the first linker, the IL-2 binding protein, the second linker, and the IL-2 protein. (Item 44) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (b) comprise, in N-to-C-terminal direction, the IL-2 protein, the first linker, the IL-2 binding protein, the second linker, and the binding moiety. (Item 45) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (c) comprise, in an N-to-C-terminal direction, the IL-2 protein, the first linker, the IL-2 binding protein, the second linker, and the affinity purification tag. (Item 46) 41. The activatable proprotein homodimer of any one of items 1 to 40, wherein the first polypeptide and the second polypeptide of (d) comprise, in an N-to-C-terminal direction, the IL-2 binding protein, the first linker, the IL-2 protein, the second linker, and the affinity purification tag. (Item 47) 47. The activatable proprotein homodimer of any one of the preceding claims, wherein the first polypeptide and the second polypeptide comprise, consist or consist essentially of an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% identical to a sequence selected from Table S4, and optionally the TEV protease cleavage site is replaced with a cleavage site cleavable by a human protease, optionally a cleavable linker selected from Table S3. (Item 48) 48. The activatable proprotein homodimer according to any one of items 1 to 47, which is substantially in homodimeric form in physiological solution or under physiological conditions, optionally under in vivo conditions. (Item 49) 49. The method of claim 1, further comprising: A recombinant nucleic acid molecule that (Item 50) 50. A vector comprising the recombinant nucleic acid molecule of item 49. (Item 51) A host cell comprising a recombinant nucleic acid molecule according to item 44 or a vector according to item 50. (Item 52) 52. A method for producing an activatable proprotein, comprising culturing the host cell of item 51 under culture conditions suitable for expression of an activatable proprotein homodimer, and isolating said activatable proprotein from the culture. (Item 53) 49. A pharmaceutical composition comprising an activatable proprotein homodimer according to any one of items 1 to 48 and a pharma- ceutically acceptable carrier. (Item 54) 54. A method for treating a disease in a subject and / or enhancing an immune response in a subject, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition according to item 53. (Item 55) 55. The method of claim 54, wherein the disease is selected from one or more of cancer, a viral infection, and an immune disorder. (Item 56) 56. The method of claim 55, wherein the cancer is a primary or metastatic cancer and is selected from one or more of melanoma (optionally metastatic melanoma), kidney cancer (optionally renal cell carcinoma), pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia (optionally lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia), multiple myeloma, lymphoma, hepatoma (hepatocellular carcinoma), sarcoma, B-cell malignancies, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary CNS lymphoma, primitive neuroectodermal tumor (medulloblastoma), bladder cancer, uterine cancer, esophageal cancer, brain cancer, head and neck cancer, cervical cancer, testicular cancer, thyroid cancer, and gastric cancer. (Item 57) 57. The method of any one of items 54 to 56, wherein after administration, the activatable proprotein homodimer is activated through cleavage by a protease in a cell or tissue, optionally a cancer cell or tissue, and said cleavage exposes the binding site(s) of the first and / or second IL-2 protein that binds to the IL-2Rβ / γc chain present on the surface of the immune cell in vitro or in vivo, thereby generating an activated protein. (Item 58) 58. The method of claim 57, wherein the activator protein binds to the IL-2Rβ / γc chain present on the surface of an immune cell in vitro or in vivo via the IL-2 protein. (Item 59) 59. The method of claim 58, wherein the immune cells are selected from one or more of a T cell, a B cell, a natural killer cell, a monocyte, and a macrophage. (Item 60) The bond between the IL-2 protein(s) and the IL-2 binding protein(s) in the activation protein (optionally a disulfide bond between the IL-2 protein(s) and the IL-2Rα protein(s)) is T reg IL-2Rα / β / γc chain expressed on T reg 60. The method of any one of claims 57 to 59, wherein the method interferes with binding of the activator protein to the (Item 61) Administration and activation of the activatable proprotein enhances the immune response in the subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 61. The method of any one of items 54 to 60, wherein the immune response is increased by about 90%, about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, and optionally wherein the immune response is an anti-cancer or anti-viral immune response. (Item 62) Administration and activation of the activatable proprotein increases cell killing in the subject by about or at least about 5%, about or at least about 10%, about or at least about 15%, about or at least about 20%, about or at least about 25%, about or at least about 30%, about or at least about 35%, about or at least about 40%, about or at least about 45%, about or at least about 50%, about or at least about 60%, about or at least about 70%, about or at least about 80%, about or at least about 90%, or 62. The method of any one of paragraphs 54 to 61, wherein the cell killing is increased by about or at least about 100%, about or at least about 200%, about or at least about 300%, about or at least about 400%, about or at least about 500%, about or at least about 600%, about or at least about 700%, about or at least about 800%, about or at least about 900%, about or at least about 1000%, about or at least about 2000%, or more, and optionally wherein the cell killing is killing of cancer cells or killing of virus infected cells. (Item 63) 56. The method of item 55, wherein the viral infection is selected from one or more of human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, hepatitis E, calicivirus associated diarrhea, rotavirus diarrhea, Haemophilus influenzae type B pneumonia and invasive disease, influenza, measles, mumps, rubella, parainfluenza associated pneumonia, respiratory syncytial virus (RSV) pneumonia, severe acute respiratory syndrome (SARS), human papillomavirus, herpes simplex type 2 genital ulcers, dengue fever, Japanese encephalitis, tick-borne encephalitis, West Nile virus associated disease, yellow fever, Epstein-Barr virus, Lassa fever, Crimean-Congo hemorrhagic fever, Ebola hemorrhagic fever, Marburg hemorrhagic fever, rabies, Rift Valley fever, smallpox, upper and lower respiratory tract infections, and poliomyelitis, and optionally wherein the subject is HIV positive. (Item 64) 56. The method of claim 55, wherein the immune disorder is selected from one or more of type I diabetes, vasculitis, and immunodeficiency. (Item 65) 65. The method according to any one of items 54 to 64, wherein the pharmaceutical composition is administered to the subject by parenteral administration. (Item 66) 66. The method of claim 65, wherein the parenteral administration is intravenous administration. (Item 67) 54. Use of the pharmaceutical composition according to item 53 in the preparation of a medicament for treating a disease in a subject and / or for enhancing an immune response in a subject. (Item 68) 54. The pharmaceutical composition according to item 53, for use in the treatment of a disease in a subject and / or for enhancing the immune response in a subject.

Claims

[Claim 1] The invention described in the specification.

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