IL-2 compositions and methods of using the same

Abstract

The present application provides 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, and other optional features, related pharmaceutical compositions, and methods of use thereof.

Description

[0001] This application is a divisional application of Chinese patent application No. 202080062791.4, entitled "IL-2 Composition and Method of Using Therewith", filed on July 10, 2020, with the corresponding PCT application filed on July 10, 2020, with the application number PCT / US2020 / 041543.

[0002] Cross-referencing of related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 908,782, filed October 1, 2019, and U.S. Provisional Application No. 62 / 873,399, filed July 12, 2019, pursuant to 35 USC § 119(e), each of which is incorporated herein by reference in its entirety.

[0004] [Declaration regarding sequence lists]

[0005] The sequence list relating to this application is provided in text form instead of a paper copy and is incorporated herein by reference. The text file containing the sequence list is named PRVA_003_02WO_ST25.txt. The text file is approximately 952KB, created on July 9, 2020, and submitted electronically via EFS-Web. [Technical Field]

[0007] This disclosure relates to an activated preprotein homodimer comprising at least two separate polypeptide chains, each chain containing an IL-2 protein, a cleavable linker and an IL-2 binding protein, and other optional features, as well as methods of use thereof. [Background Technology]

[0009] Interleukin-2 (IL-2) immunotherapy has been shown to be useful in treating cancers such as malignant melanoma and renal cell carcinoma, as well as chronic infections such as HIV infection.

[0010] However, most IL-2 therapies have certain problems. For example, current forms of IL-2 therapy have a short half-life in circulation and primarily expand immunosuppressive regulatory T cells or T cells. reg(See, for example, Arenas-Ramirez et al., Trends in Immunology. 36: 763-777, 2015). Furthermore, the effects of IL-2 therapy are primarily systemic, rather than limited to target tissues, leading to numerous serious side effects such as respiratory problems, nausea, hypotension, loss of appetite, confusion, severe infections, seizures, allergic reactions, heart problems, kidney failure, and vascular leakage syndrome. Nevertheless, IL-2 therapy can be effective, and there is an unmet need in the field to overcome these and other drawbacks.

[0011] The embodiments of this disclosure address these and more issues by providing an IL-2-containing, activatable proprotein that can be activated within diseased tissues such as cancerous tissue or tumors. [Summary of the Invention]

[0013] Embodiments of this disclosure include an activatable preprotein homodimer comprising a first polypeptide and a second polypeptide, wherein:

[0014] (a) The first and second polypeptides comprise a binding portion, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein in the N-to-C-terminal or C-to-N-terminal direction; or

[0015] (b) The first and second polypeptides contain a binding portion, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein in the N-to-C-terminal or C-to-N-terminal direction.

[0016] The binding portion of the first polypeptide binds to the binding portion of the second polypeptide, wherein the IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and wherein the IL-2 binding protein of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the binding masks the binding site of the IL-2 protein, which otherwise binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, and wherein at least one of the first or second linkers is a cleavable linker; or

[0017] (c) The first and second polypeptides contain an IL-2 protein, a first adaptor, an IL-2 binding protein, a second adaptor, and an affinity purification tag in the N-to-C-terminal or C-to-N-terminal direction; or

[0018] (d) The first and second polypeptides contain an IL-2 binding protein, a first adaptor, an IL-2 protein, a second adaptor, and an affinity purification tag in the N-to-C-terminal or C-to-N-terminal direction.

[0019] 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, wherein the binding masks the binding site of the IL-2 protein, which otherwise binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, and wherein the first linker is a cleavable linker.

[0020] In some embodiments, the first and second IL-2 proteins comprise, consist of, or are substantially composed of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to, composed of, or substantially composed of, the amino acid sequence selected from the amino acid sequence in Table S1, optionally SEQ ID NO: 1 (full-length wild-type human IL-2), amino acid sequences 21-153, and optionally include a C145X (X is any amino acid) or C145S substitution as defined in SEQ ID NO: 1. In some embodiments, the first and second IL-2 proteins comprise, consist of, or are substantially composed of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to, composed of, or substantially composed of, the amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to, composed of, or substantially composed of, the amino acid sequence in SEQ ID NO: 2 (mature human IL-2 with a C125S substitution), optionally wherein the IL-2 protein retains the S125 residue as defined in 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 in SEQ ID NO: 2.

[0021] In some embodiments, the first IL-2 protein forms a disulfide bond with the second IL-2 binding protein, and wherein the second IL-2 protein forms a disulfide bond with the first IL-2 binding protein, optionally through one or more cysteine ​​residues in claim 4 and one or more cysteine ​​residues in the first and second IL-2 binding proteins.

[0022] In some embodiments, the first and second IL-2 proteins contain one or more amino acid substitutions at positions 69, 74, and / or 128 as defined in SEQ ID NO: 2, optionally wherein the one or more amino acid substitutions are selected from V69A, Q74P, and I128T as defined in SEQ ID NO: 2. In some embodiments, the first and second IL-2 proteins contain one or more amino acid substitutions at positions 69, 74, and / or 128 as defined in SEQ ID NO: 2. IDNO:2 defines positions T3, R38, F42, Y45, E61, E62, E68, and / or L72 as containing one or more amino acid substitutions, optionally one or more of which are selected from 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 L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, including combinations thereof, optionally 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 combinations thereof with R38A, F42A, Y45A and E62A.

[0023] In some embodiments, the first and second IL-2 proteins comprise, consist of, or are substantially consist of at least 80, 85, 90, 95, 98, or 100% the same amino acid sequence as, constitutes, or is substantially consist of, SEQ ID NO: 3 (mature human IL-2 “D10” variant), wherein optionally the IL-2 protein retains any one or more of the substitutions Q74H, L80F, R81D, L85V, I86V, and / or I92F as defined in SEQ ID NO: 3.

[0024] In some embodiments, the first and second IL-2 binding proteins comprise the first and second IL-2Rα proteins, or the first and second antibodies or antigen-binding fragments thereof that specifically bind to the IL-2 protein, optionally bispecific antibodies or antigen-binding fragments thereof.

[0025] In some embodiments, the first and second IL-2Rα proteins comprise, consist of, or are substantially consist of an amino acid sequence selected from Table S2, optionally at least 80, 85, 90, 95, 98, or 100% identical to, or substantially consist of, amino acid sequences 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 as defined in SEQ ID NO: 6 (human IL-2Rα Sushi 1 to Sushi 2 domains), and / or a K38S substitution. In some embodiments, the first IL-2Rα protein forms a disulfide bond with the second IL-2 protein, and wherein the second IL-2Rα protein forms a disulfide bond with the first IL-2 protein, optionally via one or more cysteine ​​residues from claim 11 and one or more cysteine ​​residues from the IL-2 protein, optionally one or more cysteine ​​residues from claim 4, optionally selected from IL2-K35C and IL2Rα-D4C, IL2-R38C and IL2Rα-D6C, One or more cysteine ​​pairs of 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 are formed, wherein the disulfide bond binding between the IL-2 protein and the IL-2Rα protein masks the preferential binding of T. reg The binding site of the IL-2 protein on the expressed IL-2Rα / β / γc chain. In some embodiments, the first and second IL-2Rα proteins contain an alanine substitution at positions 49 and / or 68 as defined in SEQ ID NO: 6.

[0026] In some embodiments, the first and second antibodies that specifically bind to the IL-2 protein, or their antigen-binding fragments, are selected from one or more of intact antibodies, Fab, Fab', F(ab')2, monospecific Fab2, bispecific Fab2, FV, single-chain Fv (scFv), scFV-Fc, nanobodies, biantibodies, camelid antibodies, and microantibodies, optionally wherein the antibody is NARA1 or its antigen-binding fragment. In some embodiments, the binding portion of (a) and / or (b) does not bind to the IL-2 protein or IL-2-binding protein. In some embodiments, the binding portion of (a) and / or (b) binds to the IL-2 protein. In some embodiments, the binding portions of the first and second polypeptides of (a) and / or (b) are linked together by at least one non-covalent interaction, optionally dimerizing. In some embodiments, the binding portions of the first and second polypeptides of (a) and / or (b) are linked together by at least one covalent bond, optionally dimerizing. In some embodiments, the at least one covalent bond comprises at least one disulfide bond.

[0027] In some embodiments, the binding portions of the first and second polypeptides in (a) and / or (b) are selected from Table M1. In some embodiments, the binding portions of the first and second polypeptides in (a) or (b) comprise an antigen-binding domain of an immunoglobulin, including its antigen-binding fragments and variants. In some embodiments, the binding portions of the first and second polypeptides in (a) and / or (b) comprise the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3 and / or CL domains of an immunoglobulin, including their fragments and variants. In some embodiments, the binding portions of the first and second polypeptides in (a) and / or (b) comprise, in the N-to-C-terminal direction: (1) an antigen-binding domain of an immunoglobulin, including its antigen-binding fragments and variants; (2) the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3 and / or CL domains of an immunoglobulin, including their fragments and variants. In some embodiments, the antigen-binding domain comprises the VH or VL domain of an immunoglobulin, including its antigen-binding fragment and variants. In some embodiments, the binding portion of the first and second polypeptides in (a) and / or (b) does not bind an antigen. In some embodiments, the binding portion of the first and second polypeptides in (a) and / or (b) comprises the CH2CH3 domain of an immunoglobulin. In some embodiments, the immunoglobulin is derived from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM. In some embodiments, the binding portion of the first and second polypeptides in (a) and / or (b) comprises a leucine zipper peptide.

[0028] In some implementations, the affinity purification tags (c) and / or (d) are selected from multihistidine tags (optionally hexahistidine tags), VSV-G tags, universal tags, Strep-tags, S-tags, S1-tags, Phe-tags, Cys-tags, Asp-tags, Arg-tags, Myc epitope tags, KT3 epitope tags, HSV epitope tags, histidine affinity tags, hemagglutinin (HA) tags, FLAG epitope tags, E2 epitope tags, V5-tags, T7-tags, AU5 epitope tags, and AU1 epitope tags.

[0029] In some embodiments, the cleavable adapter includes a protease cleavage site, optionally wherein the cleavable adapter is selected from Table S3. In some embodiments, the protease cleavage site may be cleaved by one or more proteases selected from metalloproteinases, serine proteases, cysteine ​​proteases, and aspartic proteases. In some embodiments, the protease cleavage site may be cleaved by one or more proteases selected from MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, proteolytic enzyme, uPA, FAP, Legumain, PSA, kallikrein, cathepsin A, and cathepsin B. In some embodiments, the length of the first and / or second connector is about 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-4, or 1-3 amino acids, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0030] In some embodiments, the first connector of (a) and / or (b) is a cleavable connector, and the second connector of (a) and / or (b) is an uncleavable connector. In some embodiments, cleavage (optionally protease cleavage) of the first connector of (a) and / or (b) exposes binding sites for the first and / or second IL-2 proteins, which bind to IL-2Rβ / γc chains present on the surface of immune cells in vitro or in vivo. In some embodiments, the first connector of (a) and / or (b) is an uncleavable connector, and the second connector of (a) and / or (b) is a cleavable connector. In some embodiments, cleavage (optionally protease cleavage) of the second connector of (a) and / or (b) exposes binding sites for the first and / or second IL-2 proteins, which bind to IL-2Rβ / γc chains present on the surface of immune cells in vitro or in vivo. In some embodiments, cleavage (optionally protease cleavage) of the first linker in (c) and / or (d) exposes the binding sites of the first and / or second IL-2 proteins, which bind to the IL-2Rβ / γc chains present on the surface of immune cells in vitro or in vivo. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0031] In some embodiments, (a) the first and second peptides comprise a binding moiety, a first adapter, an IL-2 protein, a second adapter, and an IL-2 binding protein in the N-to-C-terminal direction. In some embodiments, (a) the first and second peptides comprise an IL-2 binding protein, a first adapter, an IL-2 protein, a second adapter, and a binding moiety in the N-to-C-terminal direction. In some embodiments, (b) the first and second peptides comprise a binding moiety, a first adapter, an IL-2 binding protein, a second adapter, and an IL-2 protein in the N-to-C-terminal direction. In some embodiments, (b) the first and second peptides comprise an IL-2 protein, a first adapter, an IL-2 binding protein, a second adapter, and a binding moiety in the N-to-C-terminal direction. In some embodiments, (c) the first and second peptides comprise an IL-2 protein, a first adapter, an IL-2 binding protein, a second adapter, and an affinity purification tag in the N-to-C-terminal direction. In some embodiments, (d) the first and second peptides comprise an IL-2 binding protein, a first adapter, an IL-2 protein, a second adapter, and an affinity purification tag in the N-to-C-terminal direction.

[0032] In some embodiments, the first polypeptide and the second polypeptide comprise, consist of, or are substantially consist of at least 80, 85, 90, 95, 98, or 100% the same amino acid sequence as, selected from the sequence in Table S4, optionally wherein the TEV protease cleavage site is replaced by a human protease cleavage site (optionally selected from the cleavable linkers in Table S3).

[0033] In some embodiments, the activatable preprotein is substantially in homodimer form in physiological solution or under physiological conditions, optionally under in vivo conditions.

[0034] It also includes recombinant nucleic acid molecules encoding the activated preprotein homodimers described herein, vectors containing the recombinant nucleic acid molecules described herein, and host cells containing the recombinant nucleic acid molecules or vectors described herein.

[0035] It also includes methods for generating an activatable preprotein, including culturing the host cells described herein under culture conditions suitable for expressing activatable preprotein homodimers and isolating the activatable preprotein from the culture.

[0036] It also includes pharmaceutical compositions comprising the activatable preprotein homodimer described herein and a pharmaceutically acceptable carrier.

[0037] Some implementation methods include methods for treating a disease in a subject and / or methods for enhancing an immune response in a subject, including administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject.

[0038] In some embodiments, the disease is selected from one or more of cancer, viral infection, and immune disorders. In some embodiments, the cancer is primary or metastatic cancer and is selected from one or more of the following: 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, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, 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.

[0039] In some embodiments, upon administration, the activatable preprotein homodimer is activated by protease cleavage in cells or tissues, optionally cancer cells or cancerous tissues, which exposes the binding sites of the first and / or second IL-2 proteins, which bind in vitro or in vivo to IL-2Rβ / γc chains present on the surface of immune cells, thereby producing the activated protein. In some embodiments, the activated protein binds in vitro or in vivo to IL-2Rβ / γc chains present on the surface of immune cells via IL-2 protein. In some embodiments, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages. In some embodiments, the binding between the IL-2 protein and the IL-2-binding protein in the activated protein (optionally a disulfide bond binding between the IL-2 protein and the IL-2Rα protein) masks the binding to T cells. reg The expressed IL-2Rα / β / γc chain binds to the binding site of the IL-2 protein, thereby interfering with the activation of the protein and T. reg The combination of.

[0040] In some embodiments, the administration and activation of the activatable preprotein increases the immune response in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to the control, optionally wherein the immune response is an anticancer or antiviral immune response. In some embodiments, the application and activation of the activatable preprotein increases cell killing in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more relative to the control, optionally wherein the cell killing is cancer cell killing or viral infection cell killing.

[0041] In some implementations, the viral infection is selected from one or more of the following: 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 virus 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, optionally wherein the subject is HIV positive.

[0042] In some implementations, the immune disorder is selected from one or more of type 1 diabetes, vasculitis, and immunodeficiency.

[0043] In some embodiments, the pharmaceutical composition is administered to the subject via parenteral administration. In some embodiments, parenteral administration is intravenous administration.

[0044] This also includes the use of the pharmaceutical compositions described herein in the preparation of medicaments for treating a subject's disease and / or for enhancing a subject's immune response. Specific embodiments include the pharmaceutical compositions described herein for treating a subject's disease and / or for enhancing a subject's immune response. [Attached Image Description]

[0046] Figure 1A The protein topological structures of human interleukin-2 (IL-2) and human interleukin-2 receptor α chain (IL-2Rα) are shown.

[0047] Figure 1B The quaternary structure of IL-2 complexed with its receptors IL-2Rα (CD25), IL-2Rβ (CD122), and common γ chain (CD132) is shown (PDB:2ERJ).

[0048] Figure 2A An example illustrates the fusion of the C-terminus of IL-2 with the N-terminus of IL-2Rα using a cleavable / uncleavable linker. An optional His tag is added to the C-terminus of IL-2Rα to facilitate purification. A schematic homodimeric structure is shown. IL-2 in this fusion protein cannot bind to the IL-2Rβ / γc receptor and signals through it. IL-2 activity can be restored upon protease cleavage between IL-2 and IL-2Rα.

[0049] Figure 2B Examples Figure 2AA diagram showing the protein sequence motif and conformation of the protein described in the diagram.

[0050] Figure 2C Examples illustrate the fusion of the C-terminus of Fc with the N-terminus of IL-2 using a cleavable / uncleavable linker, and the fusion of the C-terminus of IL-2 with the N-terminus of IL-2Rα using a cleavable / uncleavable linker. In these fusion proteins, IL-2 cannot bind to the IL-2Rβ / γc receptor and emit signals through it. Partial activity can be restored after protease cleavage between Fc and IL-2, while full activity can be restored after protease cleavage between IL-2 and IL-2Rα, or between IL-2 / IL-2Rα and Fc / IL-2.

[0051] Figure 2D Examples Figure 2C A diagram showing the protein sequence motifs and configurations of the proteins described in the text.

[0052] Figure 2E Examples illustrate the fusion of the C-terminus of IL-2 with the N-terminus of IL-2Rα using a cleavable / uncleavable linker, and the fusion of the C-terminus of IL-2Rα with the N-terminus of Fc using a cleavable / uncleavable linker. In these fusion proteins, IL-2 cannot bind to the IL-2Rβ / γc receptor and emit signals through it. Partial activity can be restored upon protease cleavage between Fc and IL-2, while full activity can be restored upon protease cleavage between IL-2 and IL-2Rα, or between IL-2 / IL-2Rα and Fc / IL-2Rα.

[0053] Figure 2F Examples Figure 2E A diagram showing the protein sequence motifs and configurations of the proteins described in the text.

[0054] Figure 3A An example is illustrated by the fusion of the C-terminus of IL-2Rα with the N-terminus of IL-2 using a cleavable / uncleavable linker. An optional His tag is added to the C-terminus of IL-2Rα to facilitate purification. The predicted homodimeric structure is shown. IL-2 in this fusion protein cannot bind to the IL-2Rβ / γc receptor and signals through it. IL-2 activity is restored upon protease cleavage between IL-2 and IL-2Rα.

[0055] Figure 3B Examples Figure 3A A diagram showing the protein sequence motif and conformation of the protein described in the diagram.

[0056] Figure 3CExamples illustrate the fusion of the C-terminus of Fc with the N-terminus of IL-2Rα using a cleavable / uncleavable linker, and the fusion of the C-terminus of IL-2 with the N-terminus of IL-2 using a cleavable / uncleavable linker. In these fusion proteins, IL-2 cannot bind to the IL-2Rβ / γc receptor and emit signals through it. Partial activity can be restored after protease cleavage between Fc and IL-2, while full activity can be restored after protease cleavage between IL-2 and IL-2Rα, or between IL-2 / IL-2Rα and Fc / IL-2Rα.

[0057] Figure 3D Examples Figure 3C A diagram showing the protein sequence motifs and configurations of the proteins described in the text.

[0058] Figure 3E Examples illustrate the fusion of the C-terminus of IL-2Rα with the N-terminus of IL-2 using a cleavable / uncleavable linker, and the fusion of the C-terminus of IL-2 with the N-terminus of Fc using a cleavable / uncleavable linker. In these fusion proteins, IL-2 cannot bind to the IL-2Rβ / γc receptor and emit signals through it. Partial activity can be restored upon protease cleavage between Fc and IL-2, while full activity can be restored upon protease cleavage between IL-2 and IL-2Rα, or between IL-2 / IL-2Rα and Fc / IL-2.

[0059] Figure 3F Examples Figure 3E A diagram showing the protein sequence motifs and configurations of the proteins described in the text.

[0060] Figure 4A A schematic diagram showing the activation of the “IL-2-linker-IL-2Rα-linker-His6” preprotein via protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα.

[0061] Figure 4B A schematic diagram showing the activation of the “Fc-linker-IL-2-linker-IL-2Rα” preprotein via protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα.

[0062] Figure 4C A schematic diagram is shown showing the activation of the “IL-2-linker-IL-2Rα-linker-Fc” activated preprotein by protease cleavage of the substrate linker sequence between IL-2 and IL-2Rα.

[0063] Figure 4DA schematic diagram is shown showing the activation of the “IL-2-linker-IL-2Rα-linker-Fc” activated preprotein by protease cleavage of the substrate linker sequence between IL-2 / IL-2Rα and IL-2Rα / Fc.

[0064] Figure 4E A schematic diagram showing the activation of the protease-mediated “IL-2-linker-IL-2Rα-linker-Fc” preprotein via the substrate linker sequence between IL-2Rα and Fc.

[0065] Figure 5A Examples illustrate the fusion of the C-terminus of the binding site with the N-terminus of the IL-2 protein via cleavable / uncleavable linkers, and the fusion of the C-terminus of the IL-2 protein with the N-terminus of the IL-2 binding protein via cleavable / uncleavable linkers.

[0066] Figure 5B Examples illustrate the fusion of the C-terminus of the IL-2 protein with the N-terminus of the IL-2 binding protein via cleavable / uncleavable linkers, and the fusion of the C-terminus of the IL-2 binding protein with the N-terminus of the binding site via cleavable / uncleavable linkers.

[0067] Figure 5C Examples illustrate the fusion of the C-terminus of the binding site with the N-terminus of the IL-2 binding protein via cleavable / uncleavable linkers, and the fusion of the C-terminus of the IL-2 binding protein with the N-terminus of the IL-2 protein via cleavable / uncleavable linkers.

[0068] Figure 5D Examples illustrate the fusion of the C-terminus of the IL-2 binding protein with the N-terminus of the IL-2 protein via cleavable / uncleavable linkers, and the fusion of the C-terminus of the IL-2 protein with the N-terminus of the binding site via cleavable / uncleavable linkers.

[0069] Figures 6A-6C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 6A Figure 6A shows the results of non-reducing SDS-PAGE, figure 6B shows the results of reducing SDS-PAGE, and figure 6C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 6C In the text, "1" represents the protein before TEV cleavage, and "2" represents the protein after TEV cleavage.

[0070] Figures 7A-7J Examples of representative HPLC analysis results for purified proteins are provided.

[0071] Figure 8A-8L and Figures 9A-9EExamples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0072] Figures 10A-10C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 10A Figure 10B shows the results of non-reducing SDS-PAGE, figure 10C shows the results of reducing SDS-PAGE, and figure 10C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 10C In the text, "1" represents the protein before TEV cleavage, and "2" represents the protein after TEV cleavage.

[0073] Figure 11A-11F Examples of representative HPLC analysis results for purified proteins are provided.

[0074] Figure 12A-12F Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0075] Figures 13A-13C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 13A Figure 13B shows the results of non-reducing SDS-PAGE, figure 13C shows the results of reducing SDS-PAGE, and figure 13C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 13C In the text, "1" represents the protein before uPA cleavage, and "2" represents the protein after uPA cleavage.

[0076] Figures 14A-14D Examples of representative HPLC analysis results for purified proteins are provided.

[0077] Figures 15A-15E Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0078] Figures 16A-16C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 16A Figure 16B shows the results of non-reducing SDS-PAGE, 16C shows the results of reducing SDS-PAGE, and 16C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 16CIn the text, "1" represents the protein before TEV or uPA cleavage, and "2" represents the protein after TEV or uPA cleavage. (P1773-P1778 are cleaved by TV; P1779-P1785 are cleaved by uPA).

[0079] Figures 17A-17D Examples of representative HPLC analysis results for purified proteins are provided.

[0080] Figure 18A-18N Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0081] Figures 19A-19D The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 19A Figure 19B shows the results of non-reducing SDS-PAGE, figure 19C shows the cleavage results of a single protease, and figure 19D shows the cleavage results of a dual protease. "M" in the figure represents the protein standard label. Figure 19C In the diagram, "1" represents the protein before protease cleavage, "2" represents the protein after uPA cleavage, "3" represents the protein after MMP-2 cleavage, and "4" represents the protein after proteolytic enzyme cleavage. Figure 19D In the diagram, "1" represents the protein before protease cleavage, "2" represents the protein after uPA cleavage, "3" represents the protein after MMP-2 cleavage, and "4" represents the protein after double cleavage with uPA and MMP-2.

[0082] Figure 20A-20D Examples of representative HPLC analysis results for purified proteins are provided.

[0083] Figure 21A-21Q Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0084] Figures 22A-22C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 22A Figure 22B shows the results of non-reducing SDS-PAGE, figure 22C shows the results of reducing SDS-PAGE, and figure 22C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 22C In the text, "1" represents the protein before TEV cleavage, and "2" represents the protein after TEV cleavage.

[0085] Figures 23A-23DExamples of representative HPLC analysis results for purified proteins are provided.

[0086] Figures 24A-24D Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (Cell Counting Kit-8 (CCK-8)).

[0087] Figures 25A-25C The results of SDS-PAGE of the purified protein and the cleavage of the IL-2 fusion protein are shown. Figure 25A Figure 25 shows the results of non-reducing SDS-PAGE, 25B shows the results of reducing SDS-PAGE, and 25C shows the cleavage results. "M" in the figure represents the protein standard label. Figure 25C In the diagram, "1" represents the protein before protease cleavage, "2" represents the protein after MMP-2 cleavage, "3" represents the protein after uPA cleavage, and "4" represents the protein after proteolytic enzyme cleavage.

[0088] Figures 26A-26D Examples of representative HPLC analysis results for purified proteins are provided.

[0089] Figures 27A-27D The results of SDS-PAGE and HPLC are displayed. Combined views show non-reducing SDS-PAGE results, reduced SDS-PAGE results, cleavage results, and HPLC analysis results. "M" in the figure represents a protein standard label. Figure 27C In the text, "1" represents the protein before TEV cleavage, and "2" represents the protein after TEV cleavage.

[0090] Figure 28 Examples illustrate the activity of the IL-2 fusion protein in the proliferation of M-07e, as determined by colorimetric assay (CellCounting Kit-8 (CCK-8)).

[0091] Figures 29A-29B The SDS-PAGE results of the purified protein are shown. Figure 29A Figure 29B shows the results of non-reducing SDS-PAGE, while figure 29B shows the results of reducing SDS-PAGE. "M" in the figure represents the protein standard label.

[0092] Figure 30 The results of MMP-2 cleavage are shown. "M" in the figure represents the protein standard label. "1" represents the protein before MMP-2 cleavage, and "2" represents the protein after MMP-2 cleavage.

[0093] Figure 31A-31J Examples of representative HPLC analysis results for purified proteins are provided.

[0094] Figure 32A-32M Examples illustrate the activity of IL-2 preprotein in the proliferation of M-07e, such as that determined by colorimetric assay (Cell Counting Kit 8 (CCK-8)). 【Detailed Implementation Methods】

[0096] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. While any similar or equivalent methods, materials, compositions, reagents, or cells may be used in the practice or testing of the subject matter of this disclosure, preferred methods and materials are described. All publications and references cited in this specification, including but not limited to patents and patent applications, are incorporated herein by reference in their entirety, as if each individual publication or reference were expressly and individually indicated as incorporated herein by reference for its complete exposition. Any patent application claiming priority to this application is also incorporated herein by reference in its entirety in the manner described above with respect to the disclosures and references.

[0097] Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly performed in the art or as described herein. These and related techniques and procedures can generally be performed according to conventional methods known in the art and as described in the various general and more specific references cited and discussed throughout this specification. Unless specifically defined, the nomenclature, laboratory procedures, and techniques used in relation to molecular biology, analytical chemistry, synthetic organic chemistry, and pharmaceutical and medicinal chemistry as described herein are those well-known and commonly used in the art. Standard techniques can be used in recombinant technologies, molecular biology, microbiology, chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and patient treatment.

[0098] For the purposes of this disclosure, the following terms are defined as follows.

[0099] The terms “a” and “a kind” are used in this document to refer to one or more (i.e., at least one) grammatical object. For example, “a kind of element” includes “a kind of element”, “one or more kind of element” and / or “at least one kind of element”.

[0100] "Approximately" refers to a variation of up to 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% in the reference quantity, level, value, number, frequency, percentage, size, quantity, weight, or length.

[0101] The terms “activatable preprotein,” “activatable prodrug,” “prodrug,” or “preprotein” are used interchangeably herein and refer to an activatable preprotein, or a derivative / variant thereof, comprising at least a masking portion and an active domain, as described herein. In one embodiment, the preprotein may also comprise one or more protein domains.

[0102] The term "antigen" refers to a molecule or part of a molecule that can be bound by a selective binder (e.g., an antibody) and can also be used in animals to produce antibodies capable of binding to epitopes of that antigen. An antigen may have one or more epitopes. As used herein, the term "antigen" includes substances capable of inducing an immune response to the substance under appropriate conditions and reacting with the products of the immune response. More broadly, the term "antigen" includes any substance to which an antibody binds, or any substance for which an antibody is intended, whether or not the substance is immunogenic. For such antigens, antibodies can be identified by recombinant methods, independent of any immune response.

[0103] An "antagonist" is a biological or chemical agent that interferes with or otherwise reduces the physiological effects of another agent or molecule. In some cases, antagonists specifically bind to other drugs or molecules. They include complete antagonists and partial antagonists.

[0104] An "agonist" is a biological or chemical agent that increases or enhances the physiological effects of another agent or molecule. In some cases, agonists specifically bind to other drugs or molecules. They include both full and partial agonists.

[0105] As used herein, the term "amino acid" is intended to refer to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and mimics. Naturally occurring amino acids include the 20 (L)-amino acids used 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 known to those skilled in the art, leucine, lysine, p-fluorophenylalanine, ethionine, etc. 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 portions on the amino acid or derivatization of the amino acid. Amino acid mimics include, for example, organic structures that exhibit functionally similar properties (e.g., the charge and charge space characteristics of the reference amino acid). For example, an organic structure mimicking arginine (Arg or R) would have a positively charged portion located in a similar molecular space and have the same degree of mobility as the e-amino group of the side chain of a naturally occurring Arg amino acid. The mimics also include constrained structures to maintain optimal space and charge interactions of the amino acid or amino acid functional groups. Those skilled in the art know or can determine what structures constitute functionally equivalent amino acid analogs and amino acid mimics.

[0106] As used herein, a subject “at risk of developing a disease or adverse reaction” may or may not have a detectable disease or disease symptoms, and may or may not have exhibited a detectable disease or disease symptoms prior to the treatments described herein. “At risk” means that the subject has one or more risk factors, which are measurable parameters related to disease development, as described herein and known in the art. Subjects with one or more of these risk factors are more likely to develop a disease or adverse reaction than subjects without one or more of these risk factors.

[0107] "Biocompatible" means that a material or compound will not normally impair the biological function of cells or the subject and will not cause any degree of unacceptable toxicity (including allergies and disease states).

[0108] The term "bonding" refers to the direct binding between two molecules due to interactions such as covalent bonds, electrostatic bonds, hydrophobic bonds, and ionic bonds and / or hydrogen bonds, including interactions such as salt bridges and water bridges.

[0109] "Coding sequence" refers to any nucleic acid sequence that contributes to encoding the polypeptide product of a gene. Conversely, the term "non-coding sequence" refers to any nucleic acid sequence that does not directly participate in encoding the polypeptide product of a gene.

[0110] In this disclosure, unless the context otherwise requires, the words “comprising,” “including,” and “containing” will be understood to imply inclusion of the said step or element or group of steps or elements, but do not exclude any other step or element or group of steps or elements.

[0111] "Comprising of..." means including and limited to anything that follows "comprising...". Therefore, the phrase "comprising of..." indicates that the listed element is necessary or mandatory, and no other element exists. "Substantially comprising..." means including any element listed after this phrase, and is limited to other elements that do not interfere with or contribute to the activity or function specified in this disclosure of the listed element. Therefore, the phrase "substantially comprising..." indicates that the listed element is necessary or mandatory, but other elements are optional and may or may not exist depending on whether they have a substantial impact on the activity or function of the listed element.

[0112] The terms "endotoxin-free" or "substantially endotoxin-free" generally refer to compositions, solvents, and / or containers containing at most trace amounts (e.g., amounts that have no clinically adverse physiological effects on the subject), preferably undetectable amounts of endotoxin. Endotoxins are toxins associated with certain microorganisms, such as bacteria, typically Gram-negative bacteria, although endotoxins may be present in Gram-positive bacteria, such as Listeria monocytogenes. Listeria monocytogenes The most common endotoxins are lipopolysaccharides (LPS) or lipooligosaccharides (LOS) found in the outer membranes of various Gram-negative bacteria, representing the core pathogenic characteristics of these bacteria's ability to cause disease. Small amounts of endotoxins in the human body can cause fever, lower blood pressure, activate inflammation and blood clotting, as well as other adverse physiological effects.

[0113] Therefore, in pharmaceutical manufacturing, it is often necessary to remove most or all trace amounts of endotoxins from pharmaceuticals and / or pharmaceutical containers, as even small amounts of endotoxins can have adverse effects on the human body. A depyrogenation furnace can be used for this purpose, as the decomposition of most endotoxins typically requires temperatures exceeding 300°C. For example, based on primary packaging materials (such as syringes or vials), a glass temperature of 250°C and a holding time of 30 minutes are often sufficient to reduce endotoxin levels by three logarithmic orders. Other methods for endotoxin removal have been considered, including, for example, chromatography and filtration methods, as described herein and known in the art.

[0114] Endotoxins can be detected using conventional techniques known in the art. For example, the Limulus Amoebocyte Lysate assay, which utilizes horseshoe crab blood, is a highly sensitive assay for the presence of endotoxins. In this assay, extremely low levels of LPS cause detectable coagulation of the horseshoe crab lysate due to the powerful enzyme cascade that amplifies the reaction. Endotoxins can also be quantified by enzyme-linked immunosorbent assay (ELISA). To be substantially endotoxin-free, endotoxin levels can be less than about 0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5, 1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU / mg of active compound. Typically, 1 ng of lipopolysaccharide (LPS) corresponds to about 1-10 EU.

[0115] The term "half-maximum effective concentration" or "EC" 50 "" refers to the concentration of an agent (e.g., an activatable proprotein) as described herein, at which it induces a half-maximal response between baseline and maximum after a certain number of specified exposure times; therefore, the EC50 of the gradient dose-response curve is... 50 This represents the concentration of the compound at which 50% of its maximum effect was observed. EC 50 It also represents the plasma concentration required to achieve 50% of its maximum effect in the body. Similarly, "EC" 90 "EC" refers to the concentration of a drug or composition at which 90% of its maximum effect is observed. 90 "It can be obtained from "EC" 50 The Hill slope can be calculated, or it can be determined directly from the data using conventional knowledge in the art. In some embodiments, the EC50 of the agent (e.g., an activatable proprotein) is... 50 Less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 nM. In some embodiments, the agent will have an EC of about 1 nM or less. 50 value.

[0116] "Immune response" refers to any immune response originating from the immune system, including responses from the 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, endocytosis, translocation, transport, proliferation, differentiation, activation, inhibition, cell-cell interactions, apoptosis, etc. Humoral responses include, for example, IgG, IgM, IgA, IgE responses and their corresponding effector functions.

[0117] The "half-life" of a drug, such as an activating proprotein, can refer to the time it takes for the drug to lose half of its pharmacological, physiological, or other activity, relative to its activity in the serum or tissues of an organism, or relative to any other defined point in time. "Half-life" can also refer to the time required for the amount or concentration of the drug to decrease to half of the initial amount in the serum or tissues of an organism, relative to its activity in the serum or tissues of an organism, or relative to any other defined point in time. Half-life can be measured in serum and / or any one or more selected tissues.

[0118] The terms “modulation” and “change” include “increase,” “enhancement,” or “stimulation,” as well as “decrease” or “reduction,” typically in a quantity or degree that is statistically or physiologically significant relative to a control. The amount of “increase,” “stimulation,” or “enhancement” is generally a “statistically significant” amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more times (e.g., 500, 1000 times) (including all integers and ranges such as 1.5, 1.6, 1.7, 1.8, etc.) between these values. The amount of “reduction” or “reduction” is typically a “statistically significant” amount and can include reductions of 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% (inclusive of all integers and ranges between them) produced by the absence of the composition (e.g., the absence of the reagent) or the control composition. Examples of comparative and “statistically significant” amounts are described herein.

[0119] The terms “polypeptide,” “protein,” and “peptide” are used interchangeably to refer to an amino acid polymer, not limited to any particular length. The term “enzyme” includes polypeptide or protein catalysts. This term includes modifications such as myristylation, sulfation, glycosylation, phosphorylation, and the addition or deletion of signal sequences. The terms “polypeptide” or “protein” refer to one or more chains of amino acids, each chain containing amino acids covalently linked by peptide bonds, and said polypeptide or protein may comprise multiple chains non-covalently and / or covalently linked together (by peptide bonds), having the sequence of a native protein, i.e., a protein produced by naturally occurring and, in particular, non-recombinant cells or genetically engineered or recombinant cells, and comprising a molecule having the amino acid sequence of a native protein, or a molecule having one or more amino acids of the native sequence with deletions, additions, and / or substitutions. In some embodiments, the polypeptide is a “recombinant” polypeptide produced by recombinant cells, comprising one or more recombinant DNA molecules, which are typically made from heterologous polynucleotide sequences or combinations of polynucleotide sequences that would otherwise not be found in cells.

[0120] The terms "polynucleotide" and "nucleic acid" include mRNA, RNA, cRNA, cDNA, and DNA. This term generally refers to a polymer of nucleotides at least 10 bases in length, which can be ribonucleotides or deoxynucleotides, or modified forms of any type of nucleotide. The term includes both single-stranded and double-stranded DNA. The terms "isolated DNA," "isolated polynucleotide," and "isolated nucleic acid" refer to molecules from which the total genomic DNA of a specific species has been isolated. Therefore, an isolated DNA fragment encoding a polypeptide refers to a DNA fragment containing one or more coding sequences but essentially isolated or purified from the total genomic DNA of the species from which the DNA fragment was obtained. This also includes non-coding polynucleotides that do not encode polypeptides (e.g., primers, probes, oligonucleotides). 。 It also includes recombinant vectors, such as expression vectors, viral vectors, plasmids, phage particles, bacteriophages, and viruses.

[0121] Additional coding or non-coding sequences may, but need not, be present in the polynucleotides described herein, and polynucleotides may, but need not, be linked to other molecules and / or supporting materials. Therefore, polynucleotides, or expressible polynucleotides, regardless of the length of the coding sequence itself, can be combined with other sequences, such as expression control sequences.

[0122] As used herein, the term "isolated" polypeptide or protein means that the subject protein (i) does not contain at least some other proteins normally found in nature, (ii) is substantially free of other proteins from the same source, such as from the same species, (iii) is expressed by cells from a different species, (iv) has been isolated from at least about 50% of polynucleotides, lipids, carbohydrates, or other materials (to which it is naturally associated), (v) is not associated with portions of the proteins to which the "isolated protein" is naturally associated (through covalent or non-covalent interactions), (vi) is operatively associated with polypeptides not naturally associated with it (through covalent or non-covalent interactions), or (vii) is not found in nature. Such isolated proteins may be encoded by genomic DNA, cDNA, mRNA, or other RNA, may be of synthetic origin, or any combination thereof. In some embodiments, the 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, preventative, research, or otherwise).

[0123] In some embodiments, the "purity" of any given reagent in the composition (e.g., an activatable preprotein) can be defined. For example, some compositions may contain reagents such as peptide reagents that are at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% pure (based on protein or weight-to-weight), including all decimals and ranges therebetween, measured, for example, by, but not limited to, high-performance liquid chromatography (HPLC), a well-known column chromatography method commonly used in biochemistry and analytical chemistry for the separation, identification, and quantification of compounds.

[0124] The term "reference sequence" typically refers to a nucleic acid coding sequence, or amino acid sequence, to which another sequence is being compared. All polypeptide and polynucleotide sequences described herein are included as reference sequences, including those described by name and those described in the tables and sequence listings.

[0125] Some embodiments include biologically active “variants” and “fragments” of the proteins / peptides described herein, as well as the polynucleotides encoding them. A “variant” comprises one or more substitutions, additions, deletions, and / or insertions relative to a reference polypeptide or polynucleotide (see, for example, the tables and sequence listings). The variant polypeptide or polynucleotide contains an amino acid or nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity, similarity, or homology to the reference sequence as described herein, and substantially retains the activity of the reference sequence. It also includes sequences that are different from the reference sequence and substantially retain at least one activity of the reference sequence, either 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. In some embodiments, the addition or deletion includes C-terminal and / or N-terminal addition and / or deletion.

[0126] As used herein, the term "sequence identity" or, for example, "sequence with 50% identity," refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis in a comparison within a window. Therefore, the "sequence identity percentage" can be calculated by comparing two optimally aligned sequences within a comparison window, determining the number of 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) appearing in both sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the alignment window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity produced. The optimal alignment of sequences for the comparison window can be achieved through a computerized implementation of algorithms (GAP, BESTFIT, FASTA, and TFASTA, Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or through inspection and optimal alignment (i.e., producing the highest percentage of homology within the comparison window) (generated by any chosen method). See also the BLAST family of programs, such as Altschul et al., Nucl. Acids Res. 25:3389, 1997.

[0127] The term "solubility" refers to the property of a reagent (e.g., an activatable preprotein) provided herein to dissolve in a liquid solvent and form a homogeneous solution. Solubility is typically expressed as concentration and can be described by the mass of solute per unit volume of solvent (grams of solute per kilogram of solvent, g / dL (100 mL), mg / ml, etc.), molarity, molality, mole fraction, or other similar concentrations. The maximum solute equilibrium amount 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 properties of the solvent. In some embodiments, solubility is measured at physiological pH or other pH values, 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., approximately pH 5–8). In some embodiments, solubility is measured in water or physiological buffers such as PBS or NaCl (with or without NaPO4). In specific embodiments, solubility is measured at relatively low pH (e.g., pH 6.0) and relatively high salt concentrations (e.g., 500 mM NaCl and 10 mM NaPO4). In some embodiments, solubility is measured in biological fluids (solvents) such as blood or serum. In some embodiments, the temperature may be approximately room temperature (e.g., approximately 20, 21, 22, 23, 24, 25 °C) or approximately body temperature (37 °C). In some embodiments, the solubility of the agent at room temperature or at 37 °C is at least approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, or 100 mg / ml.

[0128] The terms “subject” or “subject in need” or “patient” or “patient in need” include mammalian subjects such as human subjects.

[0129] "Substantially" or "essentially" means almost entirely or completely, for example, 95%, 96%, 97%, 98%, 99% or higher for a given quantity.

[0130] "Statistically significant" means that the result is unlikely to be a fluke. Statistical significance can be determined by any method known in the art. Commonly used significance measures include the p-value, which is the frequency or probability of the observed event occurring when the null hypothesis is true. If the obtained p-value is less than the significance level, the null hypothesis is rejected. In simple cases, the significance level is defined as a p-value of 0.05 or less.

[0131] "Therapeutic response" refers to the improvement of symptoms (whether or not it is sustained) based on the administration of one or more therapeutic agents.

[0132] As used herein, the terms “therapeutic effective amount,” “therapeutic dose,” “preventive effective amount,” or “diagnostic effective amount” refer to the amount of agent (e.g., an activated proprotein, an activated protein) required to elicit the desired biological response after administration.

[0133] As used herein, “treating” a subject (e.g., a mammal, such as a human) or cell is any type of intervention intended to alter the natural processes of an individual or cell. Treatment includes, but is not limited to, the administration of a pharmaceutical composition and may be administered preventively or after the onset of a pathological event or exposure to a pathogen. It also includes “preventive” treatment, which may be aimed at slowing the progression of the treated disease or condition, delaying the onset of the disease or condition, or reducing the severity of its onset. “Treatment” or “prevention” does not necessarily mean the complete eradication, cure, or prevention of a disease or condition or its associated symptoms.

[0134] The term "wild type" refers to the gene or gene product (e.g., polypeptide) that is most frequently observed in a population and is therefore arbitrarily designated as the "normal" or "wild type" form of the gene.

[0135] Unless otherwise expressly stated, each embodiment in this specification applies to all other embodiments.

[0136] [Active preprotein]

[0137] Embodiments of this disclosure relate to an activatable proprotein homodimer or prodrug comprising two IL-2 proteins that remain relatively inactive in their proprotein form and can be activated upon contact with a suitable environment. The activatable proproteins described herein comprise at least two separate but otherwise identical (or substantially identical) polypeptide chains linked together by non-covalent interactions and / or certain covalent bonds, such as disulfide bonds, but not by peptide or amide bonds. Typically, each polypeptide chain comprises an IL-2 protein, an IL-2 binding protein such as 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, forming a relatively stable homodimer, wherein these binding interactions block or sterically prevent the IL-2 protein in each chain from interacting with or binding to its homologous receptor on the cell (see, for example...). Figure 2A and 2C In some cases, each polypeptide chain contains a purification tag at its N- or C-terminus, which is separated from the rest of the polypeptide by a linker (see example...). Figure 2B and Figure 3BIn some cases, each polypeptide chain contains a binding domain (e.g., an Fc domain or a fragment thereof) at its N-terminus or C-terminus, which is separated from the rest of the polypeptide by a linker (see, e.g., Figures 5A-5D It binds to a binding domain on another polypeptide chain to further stabilize the preprotein homodimer (see, for example...). Figure 2C , 2E (3C and 3D). As described above, at least one linker is a cleavable linker that, upon cleavage in a target cell or tissue, restores IL-2 activity by opening the homodimer and exposing at least one active or binding site of the IL-2 protein. This allows the IL-2 portion of the now-activated protein to interact with or bind to certain of their homologous receptors on immune cells (e.g., IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains), thereby influencing downstream immune cell signaling pathways.

[0138] The activatable proprotein described in this article addresses many shortcomings of standard IL-2 therapy in treating cancer, infectious diseases, and other conditions, including high initial serum C levels that lead to overactivation of the immune system. max The 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 is due to the short PK caused by the otherwise small molecular size of IL-2 and / or the catabolism of a large number of immune cells expressing the IL-2 receptor. This leads to undesirable accumulation in target tissues (e.g., cancer, tumors) due to short PK and / or ineffective tumor targeting, as well as undesirable accumulation and immune activation in normal tissues.

[0139] Therefore, embodiments of this disclosure include an activatable preprotein homodimer (complex) comprising a first polypeptide (chain) and a second polypeptide (chain).

[0140] The first polypeptide and the second polypeptide contain a binding portion, a first linker, an IL-2 protein, a second linker, and an IL-2 binding protein in the N-to-C-terminal direction or the C-to-N-terminal direction;

[0141] Alternatively, the first and second polypeptides may contain a binding portion, a first linker, an IL-2 binding protein, a second linker, and an IL-2 protein in the N-to-C-terminal or C-to-N-terminal direction.

[0142] The binding portion of the first polypeptide binds to the binding portion of the second polypeptide, wherein the IL-2 protein of the first polypeptide binds to the IL-2 binding protein of the second polypeptide, and wherein the IL-2 binding protein of the first polypeptide binds to the IL-2 protein of the second polypeptide, wherein the (collective) binding masks the binding site of the IL-2 protein, which otherwise binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, and wherein at least one of the first or second linkers is a cleavable linker.

[0143] It also includes an activatable preprotein homodimer (complex) comprising a first polypeptide (chain) and a second polypeptide (chain).

[0144] The first and second polypeptides contain IL-2 protein, a first adaptor, an IL-2 binding protein, a second adaptor, and an optional affinity purification tag in the N-to-C-terminal or C-to-N-terminal direction;

[0145] Alternatively, the first and second polypeptides may contain an IL-2 binding protein, a first adaptor, an IL-2 protein, a second adaptor, and an optional affinity purification tag in the N-to-C-terminal or C-to-N-terminal direction.

[0146] 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, wherein the (collective) binding masks the binding site of the IL-2 protein, which otherwise binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo, and wherein the first linker is a cleavable linker.

[0147] As described above, IL-2 proteins and IL-2-binding proteins interact or bind together, for example, through non-covalent interactions or certain covalent bonds (e.g., disulfide bonds). In some cases, the binding of IL-2 proteins to IL-2-binding proteins, such as IL-2Rα protein, spatially blocks or prevents IL-2 proteins from binding to regulatory T cells (T cells). reg They bind to their homologous IL-2Rα / β / γc receptor chains expressed on [a specific protein]. In some cases, this binding and steric hindrance are preserved in the activated form of the protein and can provide [something related to immunosuppressive T cells]. reg It has advantages such as minimizing activation and reducing the consumption of pre-protein and active proteins. Exemplary IL-2 proteins and IL-2 binding proteins are described elsewhere in this document.

[0148] In some cases, the binding moieties of the first and second polypeptides dimerize together via at least one non-covalent interaction, at least one covalent bond (e.g., at least one disulfide bond), or any combination of non-covalent and covalent interactions to further stabilize the activatable preprotein and / or further mask the binding of the IL-2 protein to its homologous receptor (e.g., IL-2Rα / β / γc and / or IL-2Rβ / γc receptor chains). However, typically, the binding moieties of the first and second polypeptides are not bound together or dimerized via peptide or amide bonds. In some embodiments, the binding moieties are bound together as heterodimers (i.e., heterodimers composed of two different binding moieties). In some embodiments, the binding moieties are bound together as homodimers (i.e., homodimers composed of two identical or nearly identical binding moieties). Therefore, the binding moieties of the first and second polypeptides can be the same (or substantially the same) or different. In most cases, the binding moieties of the first and second polypeptides are the same and do not bind IL-2 protein or IL-2 binding protein. However, in some cases, one or both binding moieties may bind IL-2 protein and / or IL-2 binding protein. This article describes an exemplary combination portion.

[0149] As described above, at least one adapter contains a cleavable adapter, such as one cleavable by a protease. In some cases, one adapter contains a cleavable adapter while the other is a stable (e.g., physiologically stable) adapter. In some cases, both adapters contain cleavable adapters. In some cases, the protease is expressed in target tissues or cells, such as cancerous tissues or cancer cells. In this case, cleavage of the adapter releases the masking portion, removes the steric hindrance of the IL-2 protein, and allows selective activation of the IL-2 protein in diseased tissues or cells relative to normal or healthy tissues or cells. This selectivity and local activation not only reduces unnecessary consumption of the applied IL-2, thereby prolonging its half-life, but also enhances tissue penetration and reduces the adverse systemic effects of IL-2, among other benefits. Exemplary adapters are described herein.

[0150] In some embodiments, the homodimer binding between the first and second peptides allosterically inhibits the binding of the IL-2 protein to its targets (e.g., homologous IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains on the surface of immune cells). In these and related embodiments, the activatable preprotein exhibits no binding or substantially no binding to its target, or no more than 0.001%, 0.01%, 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or 50% binding to its target compared to binding to the active domain or the IL-2 protein alone, optionally lasting for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, or 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, or 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or longer, optionally as measured in vivo or in an in vitro target replacement assay available in the art.

[0151] The various components of each polypeptide chain can be fused in any orientation. For example, in some embodiments, the first polypeptide and the second polypeptide include a binding moiety, a first adapter, an IL-2 protein, a second adapter, and an IL-2 binding protein in the N-to-C-terminal direction. In some embodiments, the first polypeptide and the second polypeptide include an IL-2 binding protein, a first adapter, an IL-2 protein, a second adapter, and a binding moiety in the N-to-C-terminal direction. In some embodiments, the first polypeptide and the second polypeptide include a binding moiety, a first adapter, an IL-2 binding protein, a second adapter, and an IL-2 protein in the N-to-C-terminal direction. In some embodiments, the first polypeptide and the second polypeptide include an IL-2 protein, a first adapter, an IL-2 binding protein, a second adapter, and a binding moiety in the N-to-C-terminal direction. In a particular embodiment, the first polypeptide and the second polypeptide include an IL-2 protein, a first adapter, an IL-2 binding protein, a second adapter, and an affinity purification tag in the N-to-C-terminal direction. In some embodiments, (d) the first polypeptide and the second polypeptide include an IL-2 binding protein, a first adapter, an IL-2 protein, a second adapter, and an affinity purification tag in the N-to-C-terminal direction. Other possible directions will be obvious to those skilled in the art.

[0152] Some activatable preproteins consist of only two of the aforementioned protein chains; that is, they consist only of a first polypeptide and a second polypeptide, as described herein. However, in some cases, certain activatable preproteins contain multiple chains, for example, where the first and second polypeptide chains form a “core structure” on which additional or higher-order structures can be built, and these various core structures are optionally linked together by additional protein-binding domains.

[0153] This article describes the individual components of the activatable preprotein in more detail.

[0154] IL-2 protein

[0155] The activatable proproteins described herein include at least one “IL-2 protein” (or interleukin-2 protein), including human IL-2 protein. IL-2 is a cytokine signaling pathway via the IL-2 receptor (IL-2R), which consists of up to three chains called α (CD25), β (CD122), and γc (CD132). IL-2 is produced by T cells in response to antigen or mitotic stimulation and is essential for T cell proliferation and other activities crucial for regulating immune responses. IL-2 can stimulate B cells, monocytes, lymphokine-activated killer cells, natural killer cells, glioma cells, and other immune cells.

[0156] 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 in Table S1 below.

[0157] Table S1. Exemplary IL-2 peptides

[0158]

[0159] Therefore, in some embodiments, the IL-2 protein comprises, is composed of, or is substantially composed of, an amino acid sequence selected from Table S1 or an active variant or fragment thereof (having at least 80, 85, 90, 95, 98, or 100% identity with the sequence selected from Table S1). In some embodiments, the “active” IL-2 protein or fragment or variant is characterized, for example, by its ability to bind, in vitro or in vivo, to IL-2Rβ / γc and / or IL-2Rα / β / γc receptor chains present on the surface of immune cells and to stimulate downstream signaling activities, without the steric hindrance caused by the masking portion described herein. Examples of downstream signaling activities include IL-2-mediated signaling, which occurs through one or more, including combinations thereof, the JAK-STAT, PI3K / Akt / mTOR, and MAPK / ERK pathways. In summary, IL-2 signaling stimulates a range of downstream pathways, resulting in responses that play important roles in the development, function, and survival of CD4 T cells, CD8 T cells, NK cells, NKT cells, macrophages, and intestinal intraepithelial lymphocytes.

[0160] In certain embodiments, the IL-2 protein is the mature form, or active variant or fragment of IL-2, comprising, consisting of, or substantially consisting of an amino acid sequence having at least 80, 85, 90, 95, 98, or 100% identity with amino acids 21-153 of SEQ ID NO: 1. In some embodiments, the IL-2 protein comprises a C145X substitution as defined in SEQ ID NO: 1, where X is any amino acid. In certain embodiments, the protein comprises a C145S substitution as defined in SEQ ID NO: 1.

[0161] Some IL-2 proteins comprise, consist of, or are substantially composed of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to, that of SEQ ID NO:2 (mature human IL-2 with a C125S substitution). In some embodiments, the active variant or fragment of SEQ ID NO:2 retains the S125 residue, as described herein.

[0162] Relative to the exemplary amino acid sequences in Table S1, certain IL-2 proteins contain one or more defined amino acid substitutions. For example, some IL-2 proteins contain one or more amino acid substitutions selected from K35C, R38C, T41C, F42C, E61C, and V69C as defined in 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) by substituting one or more cysteine ​​residues 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 in SEQ ID NO: 2, including combinations thereof, and include, for example, one or more of these amino acid substitutions selected from V69A, Q74P, and I128T as defined in SEQ ID NO: 2. Some IL-2 proteins contain one or more amino acid substitutions, including combinations thereof, at the R38, F42, Y45, E62, E68 and / or L72 positions as defined in SEQ ID NO: 2, and include, for example, one or more amino acid substitutions selected from 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; E62A and E62L; E68A and E68V; L72A, L72G, L72S, L72T, L72Q, L72E, L72N, L72D, L72R and L72K, including combinations thereof. Specific examples include IL-2 proteins comprising 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 contain one or a combination of amino acid substitutions at T3 and / or E61 as defined in SEQ ID NO: 2, such as T3A and / or E61S. Therefore, IL-2 proteins may contain any one or more of the aforementioned amino acid substitutions, including combinations thereof.

[0163] It should be understood that any one or more of the aforementioned IL-2 proteins can be combined with any other components described herein, such as IL-2 binding proteins like IL-2Rα protein, masking portions including binding and linker portions, and other optional protein domains, to produce one or more activatable preproteins or larger multi-chain structures containing them.

[0164] IL-2 binding protein

[0165] The activatable preproteins described herein comprise at least one “IL-2 binding protein”. Examples of IL-2 binding proteins include IL-2Rα protein, including human IL-2Rα protein, and antibodies and antigen-binding fragments thereof that bind to the IL-2 protein described herein.

[0166] In a particular embodiment, the IL-2 binding protein is the human IL-2Rα protein, or a variant or fragment thereof that binds to the IL-2 protein. An exemplary human IL-2Rα amino acid sequence is provided in Table S2 below.

[0167] [Table S2. Exemplary IL-2Rα Protein]

[0168]

[0169] Therefore, in some embodiments, the IL-2Rα protein comprises, consists of, or is substantially composed of an active variant or fragment of an amino acid sequence selected from Table S2 or thereof that is at least 80, 85, 90, 95, 98, or 100% identical to a sequence selected from Table S2 and binds to the IL-2 protein. In some embodiments, the IL-2Rα protein comprises, consists of, or is substantially composed of an amino acid sequence that is at least 80, 85, 90, 95, 98, or 100% identical to amino acid 22-187 or 22-240 of SEQ ID NO: 4 (full-length wild-type human IL-2Rα).

[0170] Relative to the exemplary amino acid sequences in Table S2, certain IL-2Rα proteins contain one or more defined amino acid substitutions. For example, in some cases, IL-2Rα proteins contain one or more cysteine ​​substitutions selected from D4C, D6C, N27C, K38C, S39C, L42C, Y43C, I118C, and H120C as defined in SEQ ID NO: 6 (human IL-2Rα Sushi 1 to Sushi 2 domains). In some cases, IL-2Rα proteins contain an alanine substitution at positions 49 and / or 68 as defined in SEQ ID NO: 6. In some embodiments, IL-2Rα proteins contain a K38S substitution as defined in SEQ ID NO: 6. Therefore, IL-2Rα proteins may contain any one or more of the aforementioned amino acid substitutions, including combinations thereof.

[0171] In some 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 selected from the IL-2 protein. In a specific embodiment, the IL-2Rα and IL-2 proteins form at least one disulfide bond between 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. In a particular embodiment, as described above, the binding between the IL-2 protein and the IL-2Rα protein (e.g., disulfide bond binding) masks or spatially hinders the preferential binding of the IL-2 protein to T cells. reg The binding sites of the IL-2Rα / β / γc chains expressed on the protein. In some cases, after cleavage of at least one linker and release of the corresponding masking moiety, the active or activated form of the protein retains the binding between the IL-2 protein and the IL-2Rα protein, and therefore does not preferentially bind to T. reg The IL-2Rα / β / γc chain expressed on the surface.

[0172] As described above, in some 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 intact antibodies, Fab, Fab', F(ab')2, monospecific Fab2, bispecific Fab2, FV, single-chain Fv (scFv), scFV-Fc, nanobodies, biantibodies, camelid antibodies, and microantibodies. In specific embodiments, the antibody is NARA1 or an antigen-binding fragment thereof (see, for example, Arenas-Ramirez et al., Science Translational Medicine. 8: 367ra166, 2016; and U.S. Application No. 2019 / 0016797, which is incorporated herein by reference). In certain embodiments, similar to the above, the binding (e.g., disulfide bond binding) between the IL-2 protein and the anti-IL-2 antibody (or its antigen-binding fragment) masks or spatially impedes the preferential binding of the IL-2 protein. reg The binding sites of the IL-2Rα / β / γc chains expressed on the protein. In some cases, after cleavage of at least one linker and release of the corresponding masking moiety, the active or activated form of the protein retains the binding between the IL-2 protein and the IL-2Rα protein, and therefore does not preferentially bind to T. reg The IL-2Rα / β / γc chain expressed on the surface.

[0173] As used herein, the term "antibody" includes not only complete polyclonal or monoclonal antibodies, but also their fragments (e.g., dAb, Fab, Fab', F(ab')2, Fv), single chains (ScFv), their synthetic variants, naturally occurring variants, fusion proteins containing an antibody portion having an antigen-binding fragment with desired specificity, humanized antibodies, chimeric antibodies, and any other modified conformation of immunoglobulin molecules containing an antigen-binding site or fragment (epitope recognition site) with desired specificity. Certain characteristics and properties of antibodies (and their antigen-binding fragments) are described in more detail herein.

[0174] Antibodies or antigen-binding fragments can be virtually any type. As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding 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.

[0175] As used herein, the term "antigen-binding fragment" refers to a polypeptide fragment comprising at least one CDR of an immunoglobulin heavy chain and / or light chain that binds to a target antigen. In this respect, the antigen-binding fragment of an antibody described herein may comprise a V from an antibody that binds to a target molecule. H and V L The sequence 1, 2, 3, 4, 5 or all 6 CDRs.

[0176] The binding properties of antibodies and their antigen-binding fragments 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 its antigen-binding fragment is quantified in terms of about or in the range of about ≤10. -7 M to about 10 -8 The equilibrium dissociation constant of M is specifically bound to target molecules such as IL-2 protein or its epitopes or complexes. In some embodiments, the equilibrium dissociation constant is about or in the range of about ≤10. -9 M to approximately ≤10 -10 M. In some illustrative embodiments, the antibody or its antigen-binding fragment has an affinity (Kd or EC) of about, at least about, or less than about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM for the IL-2 protein (to which it specifically binds). 50 ).

[0177] A molecule, such as a peptide or antibody, is said to exhibit “specific binding” or “preferential binding” if it reacts or binds to a specific cell, substance, or epitope more frequently, rapidly, for a longer duration, and / or with greater affinity than it binds to other cells, substances, or epitopes. An antibody is said to “specifically bind” or “preferentially bind” to a target molecule or epitope if its binding to a target molecule or epitope has greater affinity, is easier, and / or lasts longer (e.g., in statistically significant amounts) than its binding to other substances or epitopes. Typically, one member of a pair of molecules exhibiting specific binding has a region on its surface or cavity that specifically binds and is therefore complementary to the specific spatial and / or polar organization of the other member of the molecule pair. 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 specific epitope is an antibody that binds to that specific epitope with greater affinity, is easier, and / or lasts longer than it binds to other epitopes. This definition also allows us to understand that, for example, an antibody (or part or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. The term also applies, for example, to an antibody that is specific to a particular epitope carried by many antigens, in which case the specific binding member carrying the antigen-binding fragment or domain will be able to bind to various antigens carrying the epitope; for example, it may exhibit cross-reactivity to many different forms of target antigens from multiple species sharing a common epitope.

[0178] Immunobinding generally refers to a type of non-covalent interaction that occurs between an immunoglobulin molecule and an immunoglobulin-specific antigen, such as, for example, by way of illustration and not limitation, due to electrostatic, ionic, hydrophilic and / or hydrophobic attractive or repulsive forces, steric forces, hydrogen bonds, van der Waals forces, and other interactions. The strength or affinity of an immunobinding interaction can be expressed as the dissociation constant (Kd) of the interaction, where a smaller Kd represents a greater affinity. The immunobinding properties of a selected peptide can be quantified using methods known in the art. One such method requires measuring the rates of formation and dissociation of the antigen-binding site / antigen complex, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that equally affect the rates in both directions. Thus, the “association rate constant” (Kon) and the “dissociation rate constant” (Koff) can be determined by calculating the concentration and the actual binding and dissociation rates. The ratio of Koff / Kon can be stripped of all parameters unrelated to affinity and is therefore equivalent to the dissociation constant Kd. As used herein, the term “affinity” includes the equilibrium constant for the reversible binding of two reagents, expressed as Kd or EC. 50The affinity of an antibody for the IL-2 protein or epitope can be, for example, from about 100 nanomolars (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 "affinity" refers to the resistance of a complex of two or more reagents to dissociation upon dilution.

[0179] Antibodies can be prepared using 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. For example, monoclonal antibodies specific to a target peptide can be prepared using the techniques of Kohler and Milstein, Eur. J. Immunol. 6:511-519, 1976, and modifications thereof. Methods for expressing human antibodies using transgenic animals such as mice are also included. 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. Specific examples include REGENEREX®'s VELOCIMMUNE® platform (see, for example, U.S. Patent No. 6,596,541).

[0180] In some implementations, the antibody and its antigen-binding fragment, as described herein, comprise heavy and light chain CDR sets, inserted into heavy and light chain frame region (FR) sets, respectively, which support the CDRs and define their spatial relationships relative to each other. As used herein, the term "CDR set" refers to three hypervariable regions of the V region of the heavy or light chain. 。 Starting from the N-terminus of either the heavy or light chain, these regions are designated "CDR1", "CDR2", and "CDR3", respectively. Therefore, the antigen-binding site comprises six CDRs, including a group of CDRs from each of the V regions of the heavy and light chains. Peptides containing a single CDR (e.g., CDR1, CDR2, or CDR3) are referred to herein as "molecular recognition units". Crystallographic analyses of many antigen-antibody complexes have shown that the amino acid residues of the CDRs form extensive contacts with the binding antigen, with the most extensive antigen contact being with the heavy chain CDR3. Therefore, the molecular recognition unit is primarily responsible for the specificity of the antigen-binding site.

[0181] As used herein, the term "FR group" refers to the four flanking amino acid sequences that form the CDR group of the heavy or light chain V region. Some FR residues can contact the bound antigen; however, the FRs are primarily responsible for folding the V region to the antigen-binding site, especially the FR residues directly adjacent to the CDR. Within the FR, certain amino acid residues and certain structural features are highly conserved. In this regard, all V region sequences contain an internal disulfide ring of approximately 90 amino acid residues. When the V region folds to the binding site, the CDR appears as a prominent ring motif that forms the antigen-binding surface. It is generally believed that there are conserved structural regions in the FR that influence the shape of the CDR ring folding into certain "canonical" structures—regardless of the exact CDR amino acid sequence. Furthermore, certain FR residues are known to participate in non-covalent interdomain contacts, which stabilize the interaction between the antibody heavy and light chains.

[0182] The structure and location of immunoglobulin variable domains can be determined by referring to Kabat, E.A. et al., Sequences of Proteins of Immunological Interest. 4th Edition. US Department of Health and Human Services. 1987 and its updates.

[0183] This also includes "monoclonal" antibodies, referring to a group of antibodies of the same species, where monoclonal antibodies consist of amino acids (naturally and non-naturally occurring) that participate in the selective binding of epitopes. Monoclonal antibodies are highly specific, targeting a single epitope. The term "monoclonal antibody" includes not only complete and full-length monoclonal antibodies, but also their fragments (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), their variants, fusion proteins containing antigen-binding portions, humanized monoclonal antibodies, chimeric monoclonal antibodies, and any other modified conformations of immunoglobulin molecules (which include antigen-binding fragments (epitope recognition sites) with the desired specificity and ability to bind to epitopes). It is not intended to limit the source of the antibody or the way it is prepared (e.g., by hybridoma, phage selection, recombinant expression, transgenic animals). The term includes the entire immunoglobulin as well as fragments as defined above as "antibody".

[0184] Papain, a proteolytic enzyme, preferentially cleaves IgG molecules to produce several fragments, two of which (F(ab) fragments) each contain a covalent heterodimer that includes an intact antigen-binding site. Pepsin is capable of cleaving IgG molecules to provide several fragments, including an F(ab')2 fragment containing two antigen-binding sites. Fv fragments used according to certain embodiments can be produced by preferentially proteolytically cleaving IgM, and in rare cases, by cleaving IgG or IgA immunoglobulin molecules. However, Fv fragments are more generally derived using recombinant techniques known in the art. Fv fragments comprise non-covalent VH::VL heterodimers that include antigen-binding sites that retain most of the antigen recognition and binding ability 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. o

[0185] In some implementations, single-chain Fv (scFV) antibodies are considered. For example, κ bodies can be prepared using standard molecular biology techniques following the teachings of this application for selecting antibodies with the desired specificity (Ill et al., Prot. Eng. 10:949-57, 1997); microantibodies (Martin et al., EMBO J 13:5305-9, 1994); biantibodies (Holliger et al., PNAS 90:6444-8, 1993); or Janusin (Traunecker et al., EMBO J 10:3655-59, 1991; and Traunecker et al., Int. J. Cancer Suppl. 7:51-52, 1992).

[0186] Single-chain Fv (scFv) peptides are covalently linked VH::VL heterodimers expressed by gene fusions (which include VH and VL encoding genes linked by a linker encoding the peptide). Huston et al. (PNAS USA. 85(16): 5879-5883, 1988). Various methods have been described for identifying chemical structures to convert naturally aggregated but chemically separated light and heavy polypeptide chains from the antibody V region into scFv molecules (which fold into a three-dimensional structure substantially similar to the antigen-binding site structure). 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.

[0187] In some embodiments, the antibodies or antigen-binding fragments described herein are in the form of “biantibodies.” A biantibody is a multimer of polypeptides, each polypeptide containing a first domain comprising an immunoglobulin light chain binding region and a second domain comprising an immunoglobulin heavy chain binding region. These two domains are linked (e.g., via peptide linkers) but cannot bind to each other to form an antigen-binding site: the antigen-binding site is formed by the binding of the first domain of one polypeptide in the multimer to the second domain of another polypeptide in the multimer (W094 / 13804). A biantibody fragment of an antibody consists of a VH domain (Ward et al., Nature 341:544-546, 1989). For example, biantibodies and other multivalent or multispecific fragments can be constructed by gene fusion (see W094 / 13804; and Holliger et al., PNAS USA.90:6444-6448, 1993)

[0188] This also includes microantibodies containing scFv linked to the CH3 domain (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; W094 / 13804; and Reiter et al., Nature Biotech. 14:1239-1245, 1996.

[0189] When using bispecific antibodies, these can be conventional bispecific antibodies, which can be manufactured in a variety of ways (Holliger and Winter, Current Opinion Biotechnol. 4:446-449, 1993), for example, chemically prepared or prepared from hybridomas, or can be any of the bispecific antibody fragments mentioned above. Bispecific antibodies and scFv can be constructed without the Fc region, using only the variable region, which may reduce the effectiveness against idiotype reactions.

[0190] Unlike bispecific whole antibodies, bispecific biantibodies can also be particularly useful because they can be readily constructed and expressed in *E. coli*. Phage display (WO94 / 13804) from a library can be used to easily select biantibodies (as well as many other peptides such as antibody fragments) with appropriate binding specificity. If one arm of the biantibody is to remain constant, for example, having specificity against antigen X, a library can be created where the other arm is altered and an antibody with appropriate specificity is selected. Bispecific whole antibodies can be manufactured via knots-into-holes engineering (Ridgeway et al., *Protein Eng.*, 9:616-621, 1996). o

[0191] In some embodiments, the antibody or antigen-binding fragment described herein is in the form of UniBody®. UniBody® is an IgG4 antibody with its hinge region removed (see GenMab Utrecht, The Netherlands; also see, for example, US20090226421). This antibody technology creates a stable, smaller antibody form that is expected to have a longer therapeutic window than current small antibody forms. IgG4 antibodies are considered inert and therefore do not interact with the immune system. Fully human IgG4 antibodies can be modified by eliminating the hinge region of the antibody to obtain half-molecule fragments with different stability properties relative to the corresponding full IgG4 (GenMab, Utrecht). Halving the IgG4 molecule leaves only one region on UniBody® that can bind to homologous antigens (e.g., disease targets), so UniBody® binds monovalently to only one site on the target cell. For some cancer cell surface antigens, this monovalent binding may not stimulate cancer cell growth, as seen with the use of bivalent antibodies with the same antigen specificity; therefore, UniBody® technology provides a therapeutic option for some cancer types that may be difficult to treat with conventional antibodies. The small size of UniBody® can be a significant advantage in treating certain forms of cancer, allowing molecules to be better distributed on larger solid tumors and potentially improving treatment efficacy.

[0192] In some embodiments, the antibody and antigen-binding fragments described herein are in the form of nanobodies. Microbodies are encoded by a single gene and produced in virtually all prokaryotic and eukaryotic hosts, such as *Escherichia coli* (see U.S. Patent No. 6,765,087), molds (e.g., *Aspergillus* or *Trichoderma*), and yeasts (e.g., *Saccharomyces*, *Kluyveromyces*, *Hansenula*, or *Pichia pastoris* (see U.S. Patent No. 6,838,254). The production process is scalable, and several kilograms of nanobodies have been produced. Nanobodies can be formulated as a ready-to-use solution with a long shelf life. The Nanoclone method (see WO06 / 079372) is a proprietary, automated, high-throughput B-cell-based method for generating nanobodies against desired targets.

[0193] This also includes heavy chain dimers, such as those from camel and shark antibodies. Camel and shark antibodies contain homodimer pairs of V-like and C-like domains from both chains (neither containing a light chain). Because the VH region of the heavy chain dimer IgG in camel antibodies does not hydrophobically interact with the light chain, regions in the heavy chain that normally contact the light chain become hydrophilic amino acid residues in camel antibodies. The VH domain of the heavy chain dimer IgG is called the VHH domain. Shark Ig-NAR contains a homodimer with one variable domain (called the V-NAR domain) and five C-like constant domains (C-NAR domains).

[0194] In camel antibodies, the diversity of the antibody library is determined by complementarity-determining regions (CDRs) 1, 2, and 3 in the complementary VH or VHH regions. CDR3 in the camel VHH region is characterized by its relatively long length of 16 amino acids on average (Muyldermans et al., 1994, Protein Engineering 7(9): 1129). This contrasts with the CDR3 regions of antibodies from many other species. For example, the CDR3 of mouse VH has an average length of 9 amino acids. Camel antibody-derived variable region libraries (which maintain the in vivo diversity of variable regions in camel antibodies) can be prepared by methods disclosed, for example, in U.S. Patent Application Serial No. 20050037421, published February 17, 2005.

[0195] In some embodiments, the antibody or its antigen-binding fragment is humanized. These embodiments refer to chimeric molecules typically prepared using recombinant technologies, having an antigen-binding site derived from an immunoglobulin of a non-human species, and the remaining immunoglobulin structure of the molecule being based on the structure and / or sequence of human immunoglobulins. The antigen-binding site may comprise an intact variable domain fused to a constant domain or a CDR simply transplanted to a suitable frame region within the variable domain. The epitope binding site may be wild-type or modified by substitution of one or more amino acids. This eliminates the constant region from functioning as an immunogen in human individuals, but the possibility of an immune response to the foreign variable region remains (LoBuglio et al., PNASUSA 86:4220-4224, 1989; Queen et al., PNAS USA. 86:10029-10033, 1988; Riechmann et al., Nature. 332:323-327, 1988). Illustrative methods of antibody humanization include those described in U.S. Patent No. 7,462,697.

[0196] Another approach focuses not only on providing constant regions derived from humans but also on modifying variable regions to reshape them as human-like as possible. It is well known that variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs), which vary depending on the relevant epitope and determine binding capacity, flanked by four framework regions (FRs). These framework regions are relatively conserved in a given species and are assumed to provide scaffolding for the CDRs. When preparing non-human antibodies targeting specific epitopes, the variable regions can be "remodeled" or "humanized" by grafting CDRs from non-human antibodies onto the FRs present in the human antibody to be modified. This method has been applied to various antibodies and has been documented 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 2:124T34, 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 reported. In some embodiments, the humanized antibody retains all CDR sequences (e.g., a humanized mouse antibody containing all six CDRs from a mouse antibody). In other embodiments, the humanized antibody has one or more CDRs (I, II, III, IV, V, VI) altered relative to the original antibody, which are also referred to as one or more CDRs “derived” from one or more CDRs from the original antibody.

[0197] In some embodiments, the antibody is a "chimeric" antibody. In this respect, a chimeric antibody consists of an antigen-binding fragment of an antibody operatively linked to or otherwise fused with a heterologous Fc portion of a different antibody. In some embodiments, the Fc domain or heterologous Fc domain is human-derived. In some embodiments, the Fc domain or heterologous Fc domain is mouse-derived. In other embodiments, the heterologous Fc domain may be derived from an Ig class different from the parent antibody, including IgA (including subclasses IgA1 and IgA2), IgD, IgE, IgG (including subclasses IgG1, IgG2, IgG3, and IgG4), and IgM. In a further embodiment, the heterologous Fc domain may consist of CH2 and CH3 domains derived from one or more different Ig classes. As described above, regarding humanized antibodies, the antigen-binding fragment of a chimeric antibody may contain only one or more CDRs of the antibody as described herein (e.g., 1, 2, 3, 4, 5, or 6 CDRs of the antibody described herein), or it may contain a complete variable domain (VL, VH, or both).

[0198] It will be understood that any one or more of the aforementioned IL-2 binding proteins can be combined with any other components described herein (e.g., IL-2 proteins, masking portions including binding and linker portions, and other optional protein domains) to produce one or more activatable preproteins or larger multi-chain structures containing them.

[0199] [Combined Part]

[0200] As described above, the activatable preprotein homodimer described herein comprises a first polypeptide and a second polypeptide, each containing a "binding moiety". The binding moiety promotes 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 IL-2 binding protein.

[0201] General examples of the combined parts are provided in Table M1 below.

[0202] 【Table M1. Exemplary Combination Part】

[0203]

[0204] Therefore, in some implementations, the combination portion is selected from Table M1.

[0205] In certain embodiments, the binding portion comprises an antigen-binding domain of an immunoglobulin, including its antigen-binding fragment and variants, such as a VL domain and / or a VH domain. In some embodiments, the antigen-binding domain does not bind an antigen, such as a human antigen. In some embodiments, the antigen-binding domain binds an antigen, such as a human antigen.

[0206] In some embodiments, the binding moiety comprises a constant domain of an immunoglobulin or a fragment or variant thereof. For example, in some embodiments, the binding moiety comprises the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3 and / or CL domains of an immunoglobulin, including fragments and variants thereof, and combinations thereof. In some cases, the light chain (CL) is a λ or κ chain. In some embodiments, the constant domain present in the binding moiety of the activatable preprotein homodimer provided herein is glycosylated. In some embodiments, the glycosylation is N-glycosylation. In some embodiments, the glycosylation is O-glycosylation.

[0207] In a specific embodiment, the binding portion comprises, in the N-to-C-terminal direction: (1) an antigen-binding domain of an immunoglobulin, including its antigen-binding fragments and variants; and (2) a constant domain of an immunoglobulin, including its fragments and variants, such as the CH1, CH2, CH3, CH1CH3, CH2CH3, CH1CH2CH3, and / or CL domains of an immunoglobulin, including combinations thereof. In a specific embodiment, the binding portion comprises, is composed of, or is substantially composed of the CH2CH3 domain of an immunoglobulin.

[0208] The immunoglobulin domains (antigen-binding domains, constant domains) used herein optionally include an IgG domain. However, some embodiments include alternative immunoglobulins, such as IgM, IgA, IgD, and IgE. Furthermore, all possible isotypes of various immunoglobulins are included in the current embodiments. Thus, IgG1, IgG2, IgG3, etc., are all possible molecules in the binding domain. In addition to selecting isotypes and immunoglobulin types, some embodiments also include various hinge regions (or their functional equivalents). Such hinge regions provide flexibility between the different domains of the proprotein described herein. In some embodiments, the immunoglobulin portion of the binding domain (or larger masking portion) is selected from immunoglobulin classes chosen from IgG1, IgG2, IgG3, IgG4, IgD, IgA, and IgM.

[0209] [Connector]

[0210] As described above, in some embodiments, each polypeptide contains at least one or two linkers or peptide linkers. In some embodiments, at least one linker is a cleavable linker, for example, a cleavable linker containing a protease cleavage site. In some embodiments, at least one linker is an uncleavable linker, i.e., a physiologically stable linker.

[0211] In some embodiments, the length of the first and / or second connector is approximately 1-50, 1-40, 1-30, 1-20, 1-10, 1-5, 1-4, or 1-3 amino acids, or approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. In a particular embodiment, the first connector is a cuttable connector, and the second connector is a non-cuttable connector. In some embodiments, the first connector is a non-cuttable connector, while the second connector is a cuttable connector. In some implementations, both connectors are cuttable.

[0212] In some embodiments, the cleavable linker includes 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 may be cleaved by one or more proteases selected from metalloproteinases, serine proteases, cysteine ​​proteases, and aspartic proteases. In a particular embodiment, the protease cleavage site may be cleaved by one or more proteases selected from: MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, proteolytic enzyme, uPA, FAP, Legumin, PSA, kallikrein, cathepsin A, and cathepsin B.

[0213] The following table S3 provides examples of cuttable connectors.

[0214] 【Table S3. Exemplary Cuttable Connectors】

[0215]

[0216]

[0217]

[0218] Therefore, in some embodiments, the cleavable linker is selected from Table S3. Other examples of cleavable linkers include amino acid sequences that are cleaved by serine proteases (e.g., thrombin, chymotrypsin, trypsin, elastase, kallikrein, or subtilisin). Exemplary examples of thrombin-cleavable amino acid sequences 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-. Exemplary examples of amino acid sequences that can be cleaved by elastase 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).

[0219] The cleavable linker also includes an amino acid sequence that can be cleaved by matrix metalloproteinases such as collagenase, lysosome, and gelatinase. Exemplary examples of matrix metalloproteinase-cleavable amino acid sequences 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. Exemplary examples of amino acid sequences that collagenase can cleave 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 that can be cleaved by lysosome is -Pro-Tyr-Ala-Tyr-Tyr-Met-Arg- (SEQ ID NO: 135); an example of an amino acid sequence that can be cleaved by gelatinase is -Pro-Leu-Gly-Met-Tyr-Ser-Arg- (SEQ ID NO: 136).

[0220] The cleavable linker also includes 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). The cleavable linker also includes 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.

[0221] In a particular embodiment, the cleavable linker has a half-life of about 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 18 hours, 24 hours, 36 hours, 48 ​​hours, 72 hours, or 96 hours, or any intermediate half-life, at pH 7.4, 25°C, such as at physiological pH, human body temperature (e.g., in vivo, in serum, in a given tissue).

[0222] Typically, at least one of the first or second linkers is an uncleavable linker. Exemplary uncleavable linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., PNAS USA. 83:8258-8262, 1986; U.S. Patent Nos. 4,935,233 and 4,751,180. Specific uncleavable linker sequences contain Gly, Ser, and / or Asn residues. Other near-neutral amino acids, such as Thr and Ala, may also be used in the peptide linker sequence if desired.

[0223] Some exemplary non-cuttable connectors include connectors containing Gly, Ser, and / or Asn, as shown below: [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) A linker, wherein 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 those skilled in the art.

[0224] Other examples of non-cleavable linkers include the following amino acid sequence: Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser- (SEQ ID NO: 149); Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 150); Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser-(SEQ ID NO: 151); Asp-Ala-Ala-Ala-Lys-Glu-Ala-Ala-Ala-Lys-Asp-Ala-Ala-Ala-Arg-Glu-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).

[0225] Other non-limiting examples of non-cuttable joints include DGGGS (SEQ ID NO: 154); TGEKP (SEQ ID NO: 155) (see, for example, 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); EGKSSGSGSE SKVD (SEQ ID NO: 157) (Chaudhary et al., PNAS. 87:1066-1070, 1990); KESGSVSSEQ LAQFRSLD (SEQ ID NO: 158) (Bird et al., Science. 242:423-426, 1988), GGRRGGGS (SEQ ID NO: 159); LRQRDGERP (SEQ ID NO: 160); LRQKDGGGSE RP (SEQ ID NO: 161); LRQKd(GGGS)2ERP (SEQ ID NO: 162). In a specific embodiment, the adapter comprises a Gly3 adapter sequence containing three glycine residues. In a specific embodiment, a computer program capable of modeling DNA binding sites and the peptide itself can be used (Desjarlais & Berg, PNAS. 90:2256-2260, 1993; and PNAS (91:11099-11103, 1994) or by rationally designing flexible connectors through phage display methods.

[0226] In some embodiments, the linker comprises an immunoglobulin (Ig) / antibody hinge region or a fragment thereof, for example, a hinge region obtained from or derived from an IgG1 antibody. In some embodiments, the term Ig "hinge" region refers to a polypeptide comprising an amino acid sequence having sequence identity or similarity to a portion of a naturally occurring Ig hinge region sequence, the portion optionally including cysteine ​​residues wherein disulfide bonds link the two heavy chains of the immunoglobulin. The sequence similarity of the hinge region linker of the present invention to the amino acid sequence of a naturally occurring immunoglobulin hinge region can be in the range of at least 50% to about 75-80%, and is generally greater than about 90%.

[0227] In some implementations, the adapter includes a spacer element and a cleavable element to make the cleavable element more accessible to the enzyme responsible for cleavage.

[0228] It will be understood that any one or more of the aforementioned adapters may be combined with any one or more binding moieties, IL-2 proteins, IL-2 binding proteins and / or purified tags described herein to form an activatable preprotein homodimer of the present disclosure.

[0229] [Affinity Purification Label]

[0230] In some embodiments, the first and second peptides comprise at least one affinity purification tag. Exemplary affinity purification tags include multihistidine tags (optionally a six-histidine tag), VSV-G tags (YTDIEMNRLG K; SEQ ID NO: 163), universal tags (HTTPHH; SEQ ID NO: 164), Strep-tags (WSHPQFEK; SEQ ID NO: 165 or AWAHPQPGG; SEQ ID NO: 166), S-tags (KETAAAKFER QHMDS; SEQ ID NO: 167), and S1-tags (NANNPDWDF; SEQ ID NO: 168). NO: 168), Phe-tag (composed of, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 Phe residues), Cys-tag (composed of, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 Cys residues), Asp-tag (composed of, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 Asp residues), Arg-tag (composed of, for example, about 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 Arg residues), Myc epitope tag (CEQKLISEED L, SEQ ID NO: 169), KT3 epitope tag (KPPTPPPEPE T, SEQ ID NO: 170), HSV epitope tag (QPELAPED; SEQ ID NO: 171), histidine affinity tag (KDHLIHNVHK EFHAHAHNK; SEQ ID NO: 169). (SEQ ID NO: 172), hemagglutinin (HA) tag, FLAG epitope tag (DYKDDDK; SEQ ID NO: 173), E2 epitope tag (SSTSSDFRDR; SEQ ID NO: 174), V5 tag (GKPIPNPLLG LDST; SEQ ID NO: 175), T7 tag (MASMTGGQQM G; SEQ ID NO: 176), AU5 epitope tag (TDFYLK; SEQ ID NO: 177) and AU1 epitope tag (DTYRYI; SEQ ID NO: 178).

[0231] Additional structural domains

[0232] Some activatable preproteins contain one or more additional domains, such as binding domains. In some embodiments, each polypeptide in the activatable preprotein further includes a protein domain A at one free terminal and / or a protein domain B at another free terminal.

[0233] In some embodiments, protein domains A and B may be the same or different. In a particular embodiment, protein domains A and B are selected from one or more of the following: a cell receptor targeting region (optionally a bispecific targeting region), 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, cytokines, chemokines, and soluble protein ligands.

[0234] In some implementations, one or more additional protein domains may be used to form complexes of two, three, four, five or more activatable preproteins that are linked together by the additional domains.

[0235] Table S4 below provides illustrative examples of activatable preproteins and some of their expected cleavage products (see also Examples).

[0236] [Table S4. Exemplary Activatable Preproteins]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285] Therefore, in some embodiments, the activatable preprotein comprises a first polypeptide comprising, consisting of, or substantially consisting of an amino acid sequence having at least 80, 85, 90, 95, 98, or 100% identity with sequences selected from Table S4. In some embodiments, any one or more of the aforementioned sequences (from Table S4) have protease cleavage sites (e.g., TEV protease cleavage sites) replaced with human protease cleavage sites (i.e., cleavage sites cleavable by human proteases (e.g., human proteases expressed in cancer tissue or cancer cells)) (see, for example, Table S3 of exemplary cleavable connectors).

[0286] [Usage and Pharmaceutical Composition]

[0287] Some implementation methods include methods for treating, improving symptoms of a disease or condition and / or reducing its progression in subjects in need, including administering at least one activatable proprotein to the subject, as described herein. Methods for enhancing an immune response in a subject also include administering at least one activatable proprotein to the subject, as described herein. In certain implementations, the disease is selected from one or more of cancer, viral infection, and immune disorders.

[0288] In some embodiments, upon administration, the activatable preprotein is activated by protease cleavage in cells or tissues, which releases or opens the homodimer, exposing (in vitro or in vivo) binding sites of the IL-2 protein that bind to the IL-2Rβ / γc chain present on the surface of immune cells, thereby producing the activated protein (see, for example...). Figures 4A-4D In certain embodiments, protease cleavage occurs in cancer cells or cancerous tissue, or in virus-infected cells or tissue. Typically, the activated protein possesses 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), thereby stimulating immune cells. 。In a particular implementation, the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes, and macrophages.

[0289] In some implementations, the administration and activation of the pre-activatable protein (to produce the activated protein) increases the subject's immune response relative to the control by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more. In some cases, the immune response is an anticancer or antiviral immune response. In some embodiments, the administration and activation (to produce the activated protein) of the pre-activatable protein increases cell killing in the subject by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000%, or 2000% more than the control. In some embodiments, the cell killing is cancer cell killing or viral infection cell killing.

[0290] In some implementations, the administration and activation (to produce the activated protein) of the pre-activated protein does not significantly increase the activated protein's activity on regulatory T cells (T cells). reg The binding of the IL-2Rα / β / γc chain expressed on the protein. For example, in some activated proteins, the binding between IL-2 protein and IL-2-binding proteins (e.g., the disulfide bond binding between IL-2 protein and IL-2Rα protein) is maintained after linker cleavage, masking the binding on T. reg The IL-2Rα / β / γc chain expressed on the protein binds to the IL-2 protein, thereby interfering with the binding of the activating protein to T. reg The binding. Therefore, in some embodiments, the activated protein does not significantly stimulate or enhance (T) the activatable preprotein. reg The proliferation and / or activation of ).

[0291] In some implementations, the disease is cancer, meaning the subject in need has or is suspected of having cancer. Therefore, some implementations include methods for treating, improving symptoms of cancer, or inhibiting cancer progression in the subject in need, including administering at least one activatable proprotein to the subject, as described herein. In specific implementations, the cancer is primary or metastatic cancer. In a specific implementation scheme, the cancer is selected from 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, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, breast cancer, ovarian cancer, colorectal cancer, glioma, glioblastoma multiforme, meningioma, pituitary adenoma, vestibular schwannoma, primary central nervous system 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.

[0292] In some implementations, as described above, the cancer is a metastatic cancer. In addition to the cancers described above, exemplary metastatic cancers include, but are not limited to, bladder cancer that has metastasized to the bone, liver, and / or lungs; breast cancer that has metastasized to the bone, brain, liver, and / or lungs; colorectal cancer that has metastasized to the liver, lungs, and / or peritoneum; kidney cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or lungs; lung cancer that has metastasized to the adrenal glands, bone, brain, liver, and / or other lung sites; melanoma that has metastasized to the bone, brain, liver, lungs, and / or skin / muscles; ovarian cancer that has metastasized to the liver, lungs, and / or peritoneum; pancreatic cancer that has metastasized to the liver, lungs, and / or peritoneum; prostate cancer that has metastasized to the adrenal glands, bone, liver, and / or lungs; stomach cancer that has metastasized to the liver, lungs, and / or peritoneum; thyroid cancer that has metastasized to the bone, liver, and / or lungs; uterine cancer that has metastasized to the bone, liver, lungs, peritoneum, and / or vagina; and others.

[0293] Cancer treatments can be combined with other therapies. For example, the combined therapies described herein can be administered to subjects before, during, or after other treatment interventions, including symptomatic care, radiotherapy, surgery, transplantation, hormone therapy, photodynamic therapy, antibiotic therapy, or any combination thereof. Symptomatic care includes the administration of corticosteroids to reduce cerebral edema, headache, cognitive impairment, and vomiting, and the administration of anticonvulsants to reduce seizures. Radiotherapy includes whole-brain irradiation, fractionated radiotherapy, and radiosurgery, such as stereotactic radiosurgery, which can be further combined with conventional surgery.

[0294] Some implementations therefore include combination therapies for treating cancer, including methods of treating, improving symptoms of cancer, or inhibiting cancer progression in subjects in need, including administering to a subject at least one of the activatable preproteins described herein in combination with at least one additional agent, such as a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor. In some implementations, administration of at least one activatable preprotein, relative to the additional agent alone, enhances the susceptibility of cancer to the additional agent (e.g., a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor) by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000%, or more.

[0295] Some combination therapies use one or more chemotherapeutic agents, such as small molecule chemotherapeutic agents. Non-limiting examples of chemotherapeutic agents include alkylating agents, antimetabolites, cytotoxic antibiotics, topoisomerase inhibitors (type I or II), antimicrotubule agents, etc.

[0296] Examples of alkylating agents include nitrogen mustard (e.g., methyl chloroethylamine, cyclophosphamide, nitrogen mustard, melphalan, chlorambucil, ifosfamide, and busulfan), nitrosoureas (e.g., N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotimustine, and streptozotocin), tetrazines (e.g., dacarbazine, mitozolamide, and temozolomide), aziridines (e.g., thiotepa, mitomycin, and diaziquone (AZQ)), cisplatin and its derivatives (e.g., carboplatin and oxaliplatin), and non-classical alkylating agents (optionally diprocarbazine and hexamethylmelamine).

[0297] Examples of antimetabolites include antifolate agents (e.g., methotrexate and pemetrexed), fluoropyrimidines (e.g., 5-fluorouracil and capecitabine), deoxynucleoside analogs (e.g., ancitabine, excitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelabine, cladribine, clofarabine, fludarabine, and pentostatin), and thiopurines (e.g., thioguanine and mercaptopurine).

[0298] Examples of cytotoxic antibiotics include anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, arubicin, and mitoxantrone), bleomycin, mitomycin C, mitoxantrone, and actinomycin. Examples of topoisomerase inhibitors include camptothecin, irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, neomycin, mebarone, and arubicin.

[0299] Examples of antimicrotubule agents include taxanes (such as paclitaxel and docetaxel) and vinblastine alkaloids (such as vincristine, vinblastine, vinorelbine, and vinorelbine).

[0300] Those skilled in the art will understand that the various chemotherapeutic agents described herein can be combined with any one or more of the activatable proproteins described herein and used according to any one or more methods or compositions described herein.

[0301] Some combination therapies use at least one hormone therapy agent. General examples of hormone therapy agents include hormone agonists and hormone antagonists. Specific examples of hormone agonists include progestins (progestins), corticosteroids (e.g., prednisolone, methylprednisolone, dexamethasone), insulin-like growth factor, VEGF-derived angiogenesis and lymphangiogenesis factors (e.g., VEGF-A, VEGF-A145, VEGF-A165, VEGF-C, VEGF-D, PIGF-2), fibroblast growth factor (FGF), galactoglobulin, hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, androgens, estrogens, and somatostatin analogs. Examples of hormone antagonists include hormone synthesis inhibitors, such as aromatase inhibitors, and gonadotropin-releasing hormone (GnRH) agonists (e.g., leuprorelin, goserelin, triptorelin, zurelin), including their analogs. It also includes hormone receptor antagonists, such as selective estrogen receptor modulators (SERMs; for example, tamoxifen, raloxifene, toremifene) and anti-androgens (such as flutamide, bicalutamide, nilutamide).

[0302] This also includes inhibitors of hormonal pathways, such as antibodies targeting hormone receptors. Examples include IGF receptor (e.g., IGF-IR1) inhibitors, such as cetuximab, dalotuzumab, figitumumab, ganitumab, istiratumab, and robatumumab; inhibitors of vascular endothelial growth factor receptors 1, 2, or 3 (VEGFR1, VEGFR2, or VEGFR3), such as alacizumab pegol, bevacizumab, icrucumab, and ramucirumab; inhibitors of TGF-β receptors R1, R2, and R3, such as fresolimumab and metelimumab; c-Met inhibitors, such as naxitamab; and EGF inhibitor receptors such as cetuximab, depatuxizumab, mafodotin, futuximab, imgatuzumab, and lapatuximab. emtansine, matuzumab, modotuximab, nesimumab, nimotuzumab, panitumab, tomuzotuximab, and zalutumumab; FGF receptor inhibitors such as aprutumab, ixadotin, and bemarituzumab; and PDGF receptor inhibitors such as olaratumab and tovetumab.

[0303] Those skilled in the art will understand that the various hormone therapeutic agents described herein can be combined with any one or more of the activatable proproteins described herein and used according to any one or more methods or compositions described herein.

[0304] Some combination therapies use at least one kinase inhibitor, including tyrosine kinase inhibitors. Examples of kinase inhibitors include, but are not limited to, adavosertib, afanitib, aflibercept, axitinib, bevacizumab, besutinib, cabozantinib, cetuximab, cobimetinib, crizotinib, dasatinib, entrectinib, erdafitinib, erlotinib, fostamitinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pegaptanib, ponatinib, ranitumab, regorafenib, ruxolitinib, sorafenib, sunitinib, SU6656, tofacitinib, herceptin, vandetanib, and vemuafenib.

[0305] Those skilled in the art will understand that the various kinase inhibitors described herein can be combined with any one or more of the activatable proproteins described herein and used according to any one or more methods or compositions described herein.

[0306] In some embodiments, the methods and pharmaceutical compositions described herein increase median survival in subjects by 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 weeks, or longer. In some embodiments, the methods and pharmaceutical compositions described herein increase median survival in subjects by 1 year, 2 years, 3 years, or longer. In some embodiments, the methods and pharmaceutical compositions described herein extend progression-free survival by 2, 3, 4, 5, 6, 7, 8, 9, 10 weeks, or longer. In some embodiments, the methods and pharmaceutical compositions described herein extend progression-free survival by 1 year, 2 years, 3 years, or longer.

[0307] In some embodiments, the methods and therapeutic compositions described herein are sufficient to cause tumor regression, as indicated by a statistically significant reduction in the amount of surviving tumor, for example, a reduction in tumor mass of at least 10%, 20%, 30%, 40%, 50%, or more, or by a change in scan size (e.g., a statistically significant reduction). In some embodiments, the methods and therapeutic compositions described herein are sufficient to cause disease stabilization.

[0308] In some implementations, the disease is a viral disease or viral infection. In some implementations, the viral infection is selected from one or more of the following: human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, hepatitis E, calicivirus-associated diarrhea, rotavirus diarrhea, Haemophilus influenzae type b pneumonia and invasive diseases, influenza, measles, mumps, rubella, parainfluenza-associated pneumonia, respiratory syncytial virus (RSV) pneumonia, severe acute respiratory syndrome (SARS), human papillomavirus, herpes simplex virus 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 implementation, the subject is HIV positive. In some embodiments, the methods and pharmaceutical compositions described herein enhance the antiviral immune response by about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more (relative to the control).

[0309] 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 the immune function of a subject by, for example, about or at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000% or more (relative to a control).

[0310] In some embodiments, the methods and therapeutic compositions described herein are sufficient to cause clinically relevant relief of symptoms of a specific disease indication known to a physician.

[0311] For in vivo use, as described above, for the treatment of diseases in humans or non-human mammals or for testing, the agents described herein are typically incorporated into one or more therapeutic or pharmaceutical compositions, including veterinary therapeutic compositions, prior to administration.

[0312] Therefore, some embodiments involve pharmaceutical or therapeutic compositions comprising at least one activatable proprotein, as described herein. In some cases, the pharmaceutical or therapeutic composition comprises one or more activatable proproteins as described herein, in combination with a pharmaceutically or physiologically acceptable carrier or excipient. Some pharmaceutical or therapeutic compositions further comprise at least one additional agent, such as a chemotherapeutic agent, a hormone therapy agent, and / or a kinase inhibitor as described herein.

[0313] Some therapeutic compositions contain (and some methods utilize) only one activatable preprotein. Some therapeutic compositions contain (and some methods utilize) a mixture of at least two, three, four, or five different activatable preproteins.

[0314] In a particular embodiment, the pharmaceutical or therapeutic composition comprising at least one activatable preprotein is substantially pure (protein-based or weight-based), for example, the composition has a purity of at least about 80%, 85%, 90%, 95%, 98%, or 99% (protein-based or weight-based).

[0315] In some embodiments, prior to cleavage, the first and second peptides are substantially in homodimer form in the composition or other physiological solutions or under physiological conditions such as in vivo conditions.

[0316] In some embodiments, as known in the art, the activatable preprotein described herein does not form aggregates, has desired solubility, and / or has an immunogenicity profile suitable for humans. Therefore, in some embodiments, therapeutic compositions comprising the activatable preprotein are substantially free of aggregates. For example, some compositions contain less than about 10% (by protein) of high molecular weight aggregates, or less than about 5% of high molecular weight aggregates, or less than about 4% of high molecular weight aggregates, or less than about 3% of high molecular weight aggregates, or less than about 2% of high molecular weight aggregates, or less than about 1% of high molecular weight aggregates. Some compositions contain at least about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% monodisperse activatable preprotein in terms of its apparent molecular weight.

[0317] In some embodiments, the pre-activated protein may be concentrated to about 0.1 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6, 0.7, 0.8, 0.9, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11, 12, 13, 14 or 15 mg / ml and formulated for biotherapeutic purposes.

[0318] To prepare a therapeutic or pharmaceutical composition, one or more effective or desired amounts of a pharmaceutical agent are mixed with any pharmaceutical carrier or excipient known to those skilled in the art as suitable for a particular pharmaceutical agent and / or method of administration. The pharmaceutical carrier may be liquid, semi-liquid, or solid. Solutions or suspensions for parenteral, intradermal, subcutaneous, or topical application may include, for example, sterile diluents (e.g., water), saline solutions (e.g., phosphate-buffered saline; PBS), fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antimicrobial agents (e.g., benzyl alcohol and methylparaben); antioxidants (e.g., ascorbic acid and sodium bisulfite); and chelating agents (e.g., ethylenediaminetetraacetic acid (EDTA)); and buffers (e.g., acetate, citrate, and phosphate). If administered intravenously (e.g., via intravenous infusion), suitable carriers include physiological saline or phosphate-buffered saline (PBS), and solutions containing thickeners and solubilizers, such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

[0319] The administration of the pharmaceutical agents described herein (in pure form or as a suitable therapeutic or pharmaceutical composition) can be carried out by any acceptable mode of administration for providing pharmaceutical agents for similar purposes. Therapeutic or pharmaceutical compositions can be prepared by combining a composition containing the pharmaceutical agent with a suitable physiologically acceptable carrier, diluent, or excipient, and can be formulated into formulations in solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Furthermore, other active pharmaceutical ingredients (including other small molecules described elsewhere herein) and / or suitable excipients such as salts, buffers, and stabilizers may, but are not required, be present in the composition.

[0320] Administration can be achieved through a variety of routes, including oral, parenteral, intranasal, intravenous, intradermal, intramuscular, subcutaneous, or local administration. The preferred method of administration depends on the nature of the condition to be treated or prevented. Specific implementation methods include intravenous infusion.

[0321] The carrier may include, for example, pharmaceutically or physiologically acceptable carriers, excipients, or stabilizers that are non-toxic to cells or mammals exposed thereto at the doses and concentrations used. Physiologically acceptable carriers are typically pH-buffered aqueous solutions. Examples of physiologically acceptable carriers include buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) peptides; 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 dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as polysorbate 20 (TWEEN™), polyethylene glycol (PEG), and poloxamer (PLURONICS™).

[0322] In some embodiments, one or more agents may be encapsulated in microcapsules prepared, for example, by coagulation techniques or by interfacial polymerization (e.g., hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in crude emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed., (1980). The particles or liposomes may also contain other therapeutic or diagnostic agents.

[0323] The precise dosage and duration of treatment depend on the disease being treated and can be determined empirically using known testing protocols or by testing the composition in model systems known in the art. Controlled clinical trials may also be conducted. The dosage may also vary depending on the severity of the condition to be alleviated. Pharmaceutical compositions are typically formulated and administered to exert a therapeutically useful effect while minimizing adverse side effects. The composition may be administered once or divided into several smaller doses administered at intervals. For any given subject, a specific dosage regimen may be adjusted over time according to individual needs.

[0324] Therefore, typical routes of administration of these and related therapeutic or pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, oral, rectal, vaginal, and intranasal administration. The term parenteral, as used herein, includes subcutaneous injection, intravenous, intramuscular, intrasternal injection, or infusion techniques. Therapeutic or pharmaceutical compositions according to certain embodiments of this disclosure are formulated such that the active ingredient contained therein is bioavailable when administered to a subject or patient. The composition to be administered to a subject or patient may be in the form of one or more dose units; for example, a tablet may be a single dose unit, while the container of the pharmaceutical agent described herein (in aerosol form) may contain multiple dose units. Practical methods for preparing such dosage forms are known or will be 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 to be administered typically contains a therapeutically effective amount of the pharmaceutical agent described herein for the treatment of a target disease or condition.

[0325] Therapeutic or pharmaceutical compositions can be in solid or liquid form. In one embodiment, the carrier is granular, so the composition is, for example, in tablet or powder form. The carrier can be liquid, and the composition is, for example, an oral oil, an injectable liquid, or an aerosol, which can be used for, for example, inhalation administration. When intended for oral administration, the pharmaceutical composition is preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension, and gel forms are included in forms considered solid or liquid herein. Some embodiments include sterile injectable solutions.

[0326] As a solid composition for oral administration, the pharmaceutical composition can be formulated into powders, granules, tablets, pills, capsules, chewing gum, wafers, etc. 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 carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition is in capsule form (e.g., gelatin capsules), in addition to the materials of the types described above, it may also contain a liquid carrier, such as polyethylene glycol or oil.

[0327] Therapeutic or pharmaceutical compositions may be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. As two examples, the liquid may be for oral administration or for delivery by injection. When for oral administration, preferred compositions contain one or more sweeteners, preservatives, dyes / colorants, and flavoring agents in addition to the compounds of the present invention. Compositions intended for injection administration may include one or more of surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents.

[0328] Liquid therapeutic or pharmaceutical compositions, whether in solution, suspension, or other similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixing oils, such as synthetic monoglycerides or diglycerides, which may serve as solvents or suspension media, polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and tonic agents, such as sodium chloride or glucose. Parenteral preparations may be packaged in ampoules, disposable syringes, or multi-dose vials (made of glass or plastic). Physiological saline is a preferred adjuvant. Injectable pharmaceutical compositions are preferably sterile.

[0329] Liquid therapeutic or pharmaceutical compositions intended for parenteral or oral administration shall contain an amount of the pharmaceutical agent to achieve an appropriate dosage. Typically, this amount is at least 0.01% of the target pharmaceutical agent in the composition. When intended for oral administration, this amount may be between 0.1% and 70% by weight of the composition. Some oral therapeutic or pharmaceutical compositions contain about 4% to about 75% of the target pharmaceutical agent. In some embodiments, the therapeutic or pharmaceutical composition and formulation are prepared such that the parenteral dose unit contains 0.01% to 10% (before dilution) of the target agent by weight.

[0330] Therapeutic or pharmaceutical compositions may be intended for topical application, in which case the carrier may suitably comprise a solution, emulsion, ointment, or gel matrix. For example, the matrix may contain 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 application. If intended for transdermal application, the composition may comprise a transdermal patch or an iontophoresis device.

[0331] Therapeutic or pharmaceutical compositions may be intended for rectal administration, for example, in the form of suppositories, which dissolve and release the drug in the rectum. Compositions for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0332] Therapeutic or pharmaceutical compositions may include a variety of materials that alter the physical form of solid or liquid dosage units. For example, a composition may include a material that forms a coating around an active ingredient. The material forming the coating is typically inert and may be selected from, for example, sugars, 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 include components that bind to the pharmaceutical agent to facilitate the delivery of the compound. Suitable components that can function in this way include monoclonal or polyclonal antibodies, one or more proteins, or liposomes.

[0333] Therapeutic or pharmaceutical compositions may consist essentially of dosage units and can be administered as aerosols. The term "aerosol" is used to refer to a variety of systems, from colloidal systems to systems consisting of pressurized packaging. Delivery can be by liquefying or compressing gas or by a suitable pump system that dispenses the active ingredient. Aerosols can be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. Aerosol delivery includes the necessary containers, activators, valves, sub-containers, etc., which together can form a kit. Preferred aerosols can be determined by those skilled in the art without excessive experimentation.

[0334] The compositions described herein can be prepared with a carrier that protects the agent from rapid elimination from the body, such as a timed-release formulation or a coating. Such carriers include controlled-release formulations, such as, but not limited to, implants and microencapsulated delivery systems, biodegradable biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, polyorthoester, polylactic acid, and others (known to those skilled in the art).

[0335] Therapeutic or pharmaceutical compositions can be prepared using methods known in the pharmaceutical industry. For example, a therapeutic or pharmaceutical composition intended for administration by injection may comprise one or more of a salt, buffer, and / or stabilizer, mixed with sterile distilled water to form a solution. Surfactants may be added to promote the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with a pharmaceutical agent to promote its dissolution or homogeneous suspension in an aqueous delivery system.

[0336] The therapeutic or pharmaceutical composition may be administered in a therapeutically effective amount, which will depend on a variety of factors, including the activity of the specific compound used; the metabolic stability and duration of action of the compound; the subject's age, weight, general health condition, sex, and diet; the route and timing of administration; the excretion rate; the combination of drugs; the severity of the specific disease or condition; and the subject's treatment. In some cases, the therapeutically effective daily dose (for a 70 kg mammal) is from about 0.001 mg / kg (i.e., about 0.07 mg) to about 100 mg / kg (i.e., about 7.0 g); preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 0.01 mg / kg (i.e., about 0.7 mg) to about 50 mg / kg (i.e., ~3.5 g); more preferably, the therapeutically effective dose (for a 70 kg mammal) is from about 1 mg / kg (i.e., about 70 mg) to about 25 mg / kg (i.e., about 1.75 g). In some embodiments, the therapeutically effective dose is administered weekly, bi-weekly, or monthly. In the specific implementation plan, a therapeutically effective dose is administered weekly, bi-weekly, or monthly, for example, at a dose of about 1-10 or 1-5 mg / kg, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / kg.

[0337] The combination therapies described herein may include administration of a single drug dosage form comprising an activated proprotein and an additional therapeutic agent (e.g., a chemotherapeutic agent, a hormonal therapeutic agent, a kinase inhibitor), and administration of a composition comprising an activated proprotein and an additional therapeutic agent (in its own separate drug dosage form). For example, the activated proprotein and the additional therapeutic agent may be administered to a subject together in a single oral dose composition, such as tablets or capsules, or each agent may be administered in a separate oral dosage form. Similarly, the activated proprotein and the additional therapeutic agent may be administered to a subject together in a single parenteral dose composition, such as in saline solution or other physiologically acceptable solutions, or each agent may be administered in a separate parenteral dosage form. As another example, for cell-based therapies, the activated proprotein may be mixed with cells prior to administration, may be administered as part of a single composition, or both. When using separate dose formulations, the compositions may be administered substantially at the same time, i.e., simultaneously, or at separate staggered times, i.e., sequentially and in any order; combination therapy is understood to include all of these regimens.

[0338] It also includes patient care kits containing (a) at least one activatable proprotein, as described herein; and optionally (b) at least one additional therapeutic agent (e.g., a chemotherapeutic agent, a hormone therapy agent, a kinase inhibitor). In some kits, (a) and (b) are in separate therapeutic compositions. In some kits, (a) and (b) are in the same therapeutic composition.

[0339] The kits described herein may also include one or more additional therapeutic agents or other components suitable for or desired for the treatment of an indication or for a desired diagnostic application. The kits described herein may also include one or more syringes or other components necessary for or intended to facilitate the intended mode of delivery (e.g., stents, implantable reservoirs, etc.).

[0340] In some embodiments, the patient care kit comprises separate containers, dividers, or compartments for the composition and informational material. For example, the composition may be contained in a bottle, vial, or syringe, and the informational material may be contained in conjunction with the container. In some embodiments, the individual elements of the kit are contained in a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe (with informational material attached in the form of a label). In some embodiments, the kit comprises multiple (e.g., a pack) separate containers, each containing one or more unit dosage forms (e.g., the dosage forms described herein) of the activating proprotein and optionally at least one additional therapeutic agent. For example, the kit may comprise multiple syringes, ampoules, foil packs, or blister packs, each containing a single unit dose of the activating proprotein and optionally at least one additional therapeutic agent. The kit container may be airtight, waterproof (e.g., impermeable to moisture changes or evaporation), and / or opaque.

[0341] The patient care kit optionally includes a device suitable for administering the composition, such as a syringe, inhaler, dropper (e.g., an eye dropper), swab (e.g., a cotton swab or wooden swab), or any such delivery device. In some embodiments, the device is an implantable device that dispenses a measured dose of the medication. Methods of providing the kit are also included, for example, by combining the components described herein.

[0342] [Expression and Purification System]

[0343] Some embodiments include methods and related compositions for expressing and purifying the activatable preprotein described herein. Such recombinant activatable preproteins can be conveniently prepared using, for example, the standard protocols described in Sambrook et al., (1989, ibid.), particularly Sections 16 and 17; Ausubel et al., (1994, supra.), particularly Chapters 10 and 16; and Coligan et al., Current Protocols in Protein Science (John Wiley & Sons, Inc. 1995–1997), particularly Chapters 1, 5, and 6. As a general example, an activatable preprotein can be prepared by a procedure comprising one or more of the following steps: (a) preparing one or more vectors or constructs comprising one or more polynucleotide sequences encoding individual polypeptide chains of a homodimer, which are operatively linked to one or more regulatory elements; (b) introducing one or more vectors or constructs into one or more host cells; (c) culturing one or more host cells to express the polypeptides, which bind together to form an activatable preprotein homodimer; and (d) isolating the activatable preprotein homodimer from the host cells. Alternatively, the polypeptide chains can be isolated and generated first in host cells and then incubated under suitable conditions to form an activatable preprotein homodimer.

[0344] To express the desired polypeptide, the first and / or second polypeptide chain nucleotide sequence encoding the activatable preprotein can be inserted into a suitable expression vector, i.e., a vector containing the required elements of the transcriptional and translational insertion. Expression vectors containing the sequence encoding the polypeptide of interest and suitable transcriptional and translational control elements can be constructed using methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo gene 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).

[0345] A variety of expression vector / host systems are known and can be used to contain and express polynucleotide sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage, plasmid, or copious DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with viral expression vectors (e.g., baculoviruses); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cells, and more particularly human cell systems.

[0346] The “control elements” or “regulatory sequences” present in an expression vector are those untranslated regions of the vector—enhancers, promoters, 5' and 3' untranslated regions—that interact with host cell proteins to enable transcription and translation. The strength and specificity of these elements can vary. Depending on the vector system and host used, any number of suitable transcriptional and translational elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the heterozygous lacZ promoter of the PBLUESCRIPT phagemid (Stratagene, La Jolla, CA) or PSPORT1 plasmid (GibcoBRL, Gaithersburg, MD) can be used. In mammalian cell systems, promoters derived from mammalian genes or mammalian viruses are generally preferred. If it is necessary to generate cell lines containing multiple copies of sequences encoding polypeptides, SV40- or EBV-based vectors (used with appropriate selection markers) can be advantageously used.

[0347] In bacterial systems, a variety of expression vectors can be selected based on the intended use of the expressed peptide. For example, when large quantities are required, vectors that guide the high-level expression of easily purified fusion proteins can be used. Such vectors include, but are not limited to, multifunctional E. coli cloning and expression vectors, such as BLUESCRIPT (Stratagene), in which the sequence encoding the target peptide is linked to the vector by a reading frame with the sequence of the N-terminal Met and the subsequent seven β-galactosidase residues, thereby producing a hybrid protein; pIN vector (Van Heeke & Schuster, J. Biol. Chem. 264:5503 5509 (1989)); etc. pGEX vector (Promega, Madison, Wis) can also be used to express exogenous peptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption onto glutathione-agarose beads and then elution in the presence of free glutathione. Proteins prepared in such systems can be engineered to include cleavage sites for heparin, thrombin, or factor XA protease (so that the target clonal polypeptide can be released from the GST portion).

[0348] Some implementations employ *E. coli*-based expression systems (see, e.g., Structural Genomics Consortium et al., *Nature Methods*, 5:135-146, 2008). These and related implementations may rely partially or entirely on ligation-independent cloning (LIC) to generate suitable expression vectors. In certain implementations, protein expression may be controlled by T7 RNA polymerases (e.g., the pET vector family). These and related implementations may utilize the expression host strain BL21(DE3), a λDE3 lysogen of BL21, which supports T7-mediated expression and lacks the lon and ompT proteases used to enhance the stability of the target protein. Also included are expression host strains carrying plasmids encoding tRNAs rarely used in *E. coli*, such as *ROSETTA*. ™ (DE3) and Rosetta 2 (DE3) strains. Using reagents marketed under the trademarks BENZONASE® nucleases and BUGBUSTER® protein extraction reagents can also improve cell lysis and sample handling. For cell culture, self-inducible media can enhance the efficiency of many expression systems, including high-throughput expression systems. This type of medium (e.g., the OVERNIGHT EXPRESS™ self-inducible system) gradually induces protein expression through metabolic shifts without the need for artificial inducers such as IPTG. Specific implementations use hexahistine tags (e.g., marketed under the trademark HIS...). The process involves TAG® fusion (those sold commercially), followed by immobilized metal affinity chromatography (IMAC) purification or related techniques. However, in some respects, clinical-grade proteins can be isolated from *E. coli* inclusion bodies without or without affinity tags (see, for example, Shimp et al., *ProteinExpr Purif.* 50:58-67, 2006). As a further example, some implementations can employ cold-stress-induced high-yield *E. coli* production systems, as overexpression of proteins in *E. coli* at low temperatures enhances their solubility and stability (see, for example, Qing et al., *Nature Biotechnology.* 22:877-882, 2004).

[0349] This also includes high-density bacterial fermentation systems. For example, high-density culture of alkali-producing bacteria allows for protein production at cell densities exceeding 150 g / L and expression of recombinant proteins at titers exceeding 10 g / L.

[0350] In the yeast *Saccharomyces cerevisiae*, many vectors containing constitutive or inducible promoters, such as α-factors, alcohol oxidases, and PGH, can be used. For reviews, see Ausubel et al. (ibid.) and Grant et al., *Methods Enzymol.* 153:516-544 (1987). Also included are the *Pichia pastoris* pandoris expression systems (see, for example, Li et al., *Nature Biotechnology.* 24, 210–215, 2006; and Hamilton et al., *Science.* 301:1244, 2003). Some implementations include yeast systems engineered to selectively glycosylate proteins, including yeasts with humanized N-glycosylation pathways (see, for example, 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 fermenters of 15L, 50L, 100L, and 200L.

[0351] When using plant expression vectors, expression of the sequence encoding the polypeptide can be driven by any of a number of promoters. For example, viral promoters such as the 35S and 19S promoters of CaMV can be used alone or in combination with an Ω leader sequence from TMV (Takamatsu, EMBO J.6:307-311 (1987)). Alternatively, plant promoters such as the small subunit of RUBISCO or the heat shock promoter 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 have been described in many widely available reviews (see, for example, Hobbs in McGraw Hill, Yearbook of Science and Technology, pp. 191-196 (1992)).

[0352] Insect systems can also be used to express target peptides. For example, in one such system, the Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express a foreign gene in *Ardisia crenata* or *Ardisia japonica* cells. The sequence encoding the peptide can be cloned into a non-essential region of the virus, such as the polyhedrosis gene, and placed under the control of a polyhedrosis promoter. Successful insertion of the peptide-encoding sequence will inactivate the polyhedrosis gene and produce a recombinant virus lacking the coat protein. The recombinant virus can then be used to infect, for example, *Ardisia crenata* or *Ardisia japonica* cells in which the target peptide can be expressed (Engelhard et al., Proc. Natl. Acad. Sci. USA 91:3224-3227 (1994)). Baculovirus expression systems are also included, including those utilizing SF9, SF21, and *T. ni* cells (see, e.g., Murphy and Piwnica-Worms, Curr Protoc Protein Sci. Chapter 5: Unit 5.4, 2001). Insect systems can provide post-translational modifications similar to those of mammalian systems.

[0353] In mammalian host cells, a number of virus-based expression systems are commonly available. For example, when adenovirus is used as an expression vector, the sequence encoding the target polypeptide can be linked to an adenoviral transcription / translation complex consisting of a late promoter and a triple leader sequence. Insertion of a non-essential viral genome E1 or E3 region can be used to obtain a live virus capable of expressing the polypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad. Sci. USA 81:3655-3659 (1984)). Furthermore, transcriptional enhancers, such as Rous sarcoma virus (RSV) enhancers, can be used to increase expression in mammalian host cells.

[0354] Examples of useful mammalian host cell lines include monkey kidney CV1 cell lines transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cell subclones for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); young hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCCCCL70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); and buffalo rat hepatocytes (BRL 3A, ATCC CRL 1651). Human lung cells (W138, ATCCCCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and human liver cancer cell line (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 reviews of certain mammalian host cell lines suitable for protein production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.KC Lo, ed., HumanaPress, Totowa, NJ, 2003), pp. 255-268. Some preferred mammalian cell expression systems include expression systems based on CHO and HEK293 cells. Mammalian expression systems can utilize attached cell lines, for example, in T-flasks, roller flasks, or cell factories, or in suspension cultures, for example, in 1L and 5L rotary tanks, 5L, 14L, 40L, 100L, and 200L stirred tank bioreactors, or 20 / 50L and 100 / 200L WAVE bioreactors, as well as others known in the art.

[0355] This also includes cell-free protein expression. These and related implementations typically utilize purified RNA polymerases, ribosomes, tRNA, and ribonucleotides; these reagents can be produced by extraction from cells or cell-based expression systems.

[0356] Specific initiation signals can also be used to achieve more efficient translation of sequences encoding the target polypeptide. Such signals include the ATG start codon and adjacent sequences. When the sequence encoding the polypeptide, its start codon, and the upstream sequence are inserted into an appropriate expression vector, no additional transcriptional or translational control signals are required. However, when only the coding sequence or a portion thereof is inserted, exogenous translational control signals, including the ATG start codon, should be provided. Furthermore, the start codon should be in the correct reading frame to ensure translation of the entire inserted fragment. Exogenous translational elements and start codons can be of various sources, natural and synthetic. Expression efficiency can be enhanced by including enhancers suitable for the specific cell system used, such as those described in the literature (Scharf. et al., Results Probl. Cell Differ. 20:125-162 (1994)).

[0357] Furthermore, host cell lines can be selected based on their ability to regulate the expression of the inserted sequence or to process the expressed protein in the desired manner. Such modifications to peptides include, but are not limited to, post-translational modifications such as acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. Post-translational processing of proteins by cleaving the “prepro” form can also be used to facilitate proper insertion, folding, and / or function. Different host cells, such as yeast, CHO, HeLa, MDCK, HEK293, and W138 (excluding bacterial cells), with or even lacking specific cellular mechanisms and characteristic mechanisms (for such post-translational activities), can be selected to ensure proper modification and processing of exogenous proteins.

[0358] For long-term, high-yield production of recombinant proteins, stable expression is generally preferred. For example, cell lines stably expressing the target polynucleotide can be transformed using expression vectors containing viral origins of replication and / or endogenous expression elements and selection marker genes (located on the same or different vectors). After vector introduction, cells can be grown in enrichment media for approximately 1–2 days before being converted to selective media. The purpose of the selection marker is to confer selective resistance; its presence allows for the growth and recovery of cells that successfully express the introduced sequence. Tissue culture techniques suitable for the cell type can be used to proliferate resistant clones of stably transformed cells. Transient production can also be employed, for example, through transient transfection or infection. Exemplary mammalian expression systems suitable for transient production include HEK293 and CHO-based systems.

[0359] Any number of selection systems can be used to recover transformed or transduced cell lines. These include, but are not limited to, the genes for herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977)) and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823 (1990)), which can be used for tk- or aprt- cells, respectively. In addition, resistance to antimetabolites, antibiotics, or herbicides can be used as a basis for selection; for example, dhfr (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567-70 (1980)) confers resistance to methotrexate; npt (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)) confers resistance to aminoglycosides, neomycin, and G-418; and als or pat (Murry, ibid.) confers resistance to chlorsulfuron and phosphinotricin acetyltransferases, respectively. Other selectable genes have been described, such as trpB, which allows cells to use indole instead of tryptophan, or hisD, which allows cells to use histinol instead of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047-51 (1988)). The use of such markers is widespread, including green fluorescent protein (GFP) and other fluorescent proteins (such as RFP, YFP), anthocyanins, β-glucuronidase and its substrate GUS, luciferase and its substrate luciferin. They are not only widely used to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a particular vector system (see, for example, Rhodes et al., Methods Mol. Biol. 55:121-131 (1995)).

[0360] This also includes high-throughput protein production systems or micro-production systems. Certain aspects can be utilized, for example, for hexahistine fusion tags for protein expression and purification on metal-chelate modified slides or MagneHisNi-Particles (see, e.g., Kwon et al., BMC Biotechnol. 9:72, 2009; and Lin et al., Methods MolBiol. 498:129-41, 2009). High-throughput cell-free protein expression systems are also included (see, e.g., Sitaraman et al., Methods Mol Biol. 498:229-44, 2009).

[0361] Various protocols for detecting and measuring the expression of polynucleotide-encoded products, using conjugates or antibodies (e.g., product-specific polyclonal or monoclonal antibodies), are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), Western blotting, radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). These and other analyses are described in Hampton et al., Serological Methods, a Laboratory Manual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983) and elsewhere.

[0362] Various labeling and conjugation techniques are known to those skilled in the art and can be used for a wide range of nucleic acid and amino acid assays. Methods for generating labeled hybridization or PCR probes for detecting sequences associated with polynucleotides include oligolabeling, nick translation, end labeling, or PCR amplification (using labeled nucleotides). Alternatively, the sequence or any portion thereof can be cloned into a vector to generate mRNA probes. Such vectors are known in the art, commercially available, and can 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 can be performed using a variety of commercially available kits. Suitable reporter molecules or labels that can be used include radionuclides, enzymes, fluorescent agents, chemiluminescent agents or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, etc.

[0363] Host cells transformed with one or more target polynucleotide sequences can be cultured under conditions suitable for protein expression and recovery (from cell cultures). Certain specific embodiments utilize serum-free cell expression systems. Examples include HEK293 cells and CHO cells, which can be grown on serum-free media (see, for example, Rosser et al., ProteinExpr. Purif. 40:237–43, 2005; and U.S. Patent No. 6,210,922).

[0364] Depending on the sequence and / or the vector used, the recombinant cell-generated activatable proprotein can be secreted or contained within the cell. As those skilled in the art will understand, expression vectors containing polynucleotides can be engineered to contain a signal sequence that directs the secretion of the encoded polypeptide across the membrane of a prokaryotic or eukaryotic cell. Other recombinant constructs can be used to link the sequence encoding the target polypeptide to a nucleotide sequence encoding a polypeptide domain, which can facilitate the purification and / or detection of soluble proteins. Examples of such domains include cleavable and cleavable affinity purification and epitope tags, such as avidin, FLAG tags, multihistidine tags (e.g., 6xHis), cMyc tags, V5 tags, glutathione S-transferase (GST) tags, etc.

[0365] Proteins produced by recombinant cells can be purified and characterized using a variety of techniques known in the art. Exemplary systems for protein purification and analysis of protein purity include rapid protein liquid chromatography (FPLC) (e.g., AKTA and Bio-Rad FPLC systems) and high-performance liquid chromatography (HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemical methods for purification include ion exchange chromatography (e.g., Q, S), size exclusion chromatography, salt gradient, affinity purification (e.g., Ni, Co, FLAG, maltose, glutathione, protein A / G), gel filtration, reversed phase, ceramic HYPERD® ion exchange columns and hydrophobic interaction columns (HIC), and others known in the art. Analytical methods such as SDS-PAGE (e.g., Coomassie, silver staining), immunoblotting, Bradford, and ELISA are also included, which can be used at any step of the production or purification process and are typically used to measure the purity of protein compositions.

[0366] It also includes methods for concentrating activatable preproteins and compositions comprising concentrated soluble activatable preproteins. In some aspects, such concentrated solutions of at least one activatable preprotein contain protein concentrations of about or at least about 5 mg / mL, 8 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, or higher.

[0367] In some respects, such compositions can be substantially monodisperse, meaning that when determined, for example by size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation, the activatable preprotein is primarily (i.e., at least about 90% or more) present in a distinct molecular weight form.

[0368] In some respects, such compositions have a purity of at least about 90% (based on protein), or in some respects, at least about 95% purity, or in some embodiments, at least 98% purity. Purity can be determined by any conventional analytical method known in the art.

[0369] In some aspects, the content of high molecular weight aggregates in such compositions is less than about 10% (compared to the total amount of protein present), or in some embodiments, the content of high molecular weight aggregates in such compositions is less than about 5%, or in some aspects, the content of high molecular weight aggregates in such compositions is less than about 3%, or in some embodiments, the content of high molecular weight aggregates is less than about 1%. The content of high molecular weight aggregates can be determined by a variety of analytical techniques, including, for example, size exclusion chromatography, dynamic light scattering, or analytical ultracentrifugation.

[0370] Examples of concentration methods considered in this article include lyophilization, which is commonly used when the solution contains small amounts of soluble components that are not the target protein. Lyophilization is typically performed after an HPLC run and can remove most or all of the volatile components from the mixture. Ultrafiltration is also included, which typically uses one or more selectively permeable membranes to concentrate protein solutions. Membranes allow water and small molecules to pass through while retaining proteins; the solution can be pressed onto the membrane using techniques such as mechanical pumps, pneumatic pressure, or centrifugation.

[0371] In some embodiments, the activatable preprotein in the composition has a purity of at least about 90%, as measured according to conventional techniques in the art. In some embodiments, such as diagnostic compositions or certain pharmaceutical or therapeutic compositions, the activatable preprotein composition has a purity of at least about 95%, or at least about 97%, 98%, or 99%. In some embodiments, such as when used as a reference or research reagent, the activatable preprotein may have a lower purity and may have a purity of at least about 50%, 60%, 70%, or 80%. Purity can be measured holistically or in relation to selected components, such as other proteins, for example, protein-based purity.

[0372] Purified, activatable proproteins can also be characterized based on their biological properties. Binding affinity and binding kinetics can be measured using a variety of techniques known in the art, such as Biacore® and related techniques utilizing surface plasmon resonance (SPR), an optical phenomenon capable of detecting unlabeled interactants in real time. SPR-based biosensors can be used to determine activity concentrations, screen, and characterize activity in terms of affinity and kinetics. The presence or level of one or more biological activities can be measured using cell-based assays, including those utilizing at least one IL-2 receptor, optionally functionally coupled to a reading or indicator (e.g., a fluorescent or luminescent indicator of biological activity), as described herein.

[0373] In some embodiments, as described above, the activatable preprotein composition is substantially endotoxin-free, including, for example, about 95% endotoxin-free, preferably about 99% endotoxin-free, and more preferably about 99.99% endotoxin-free. The presence of endotoxins can be detected using conventional techniques in the art, as described herein. In specific embodiments, the activatable preprotein composition is prepared from eukaryotic cells such as mammalian or human cells (in a substantially serum-free culture medium). In some embodiments, as described herein, the endotoxin content of the activatable preprotein composition is less than about 10 EU / mg of activatable preprotein, or less than about 5 EU / mg of activatable preprotein, less than about 3 EU / mg of activatable preprotein, or less than about 1 EU / mg of activatable preprotein.

[0374] In some embodiments, the activatable preprotein composition comprises less than about 10% wt / wt of high molecular weight aggregates, or less than about 5% wt / wt of high molecular weight aggregates, or less than about 2% wt / wt of high molecular weight aggregates, or less than about 1% wt / wt of high molecular weight aggregates.

[0375] This also includes protein-based analytical assays and methods for assessing characteristics such as protein purity, size, solubility, and aggregation. Protein purity can be assessed in a variety 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-terminal and C-terminal sequencing and peptide-mapping (see, for example, Allen et al., Biologicals. 24:255-275, 1996). Examples of methods for evaluating higher-order structures include circular dichroism (see, for example, Kelly et al., Biochim Biophys Acta. 1751:119-139, 2005), fluorescence spectroscopy (see, for example, Meagher et al., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide-deuterium exchange kinetics, differential scanning calorimetry, NMR spectroscopy, and immunoreactivity with conformation-sensitive antibodies. Higher-order structures can also be evaluated as a function of various parameters such as pH, temperature, or added salts. Examples of methods for evaluating 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 evaluated, for example, by reversed-phase HPLC and hydrophobic interaction chromatography HPLC. Glycosylation can affect pharmacokinetics (e.g., clearance), conformation or stability, receptor binding and protein function, and can be assessed by, for example, mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy.

[0376] As described above, some embodiments include the use of SEC-HPLC to assess protein characteristics such as purity, size (e.g., size homogeneity), or degree of aggregation, and / or to purify proteins. SEC, also including 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 according to their size (or more specifically, their hydrodynamic volume), diffusion coefficient, and / or surface properties. The process is generally used to separate biomolecules, determine molecular weight, and determine the molecular weight distribution of polymers. Typically, biological or protein samples (e.g., protein extracts produced according to protein expression methods provided herein and known in the art) are loaded into size exclusion columns of selected sizes having a defined stationary phase (porous material) (preferably a phase that does not interact with the proteins in the sample). In some aspects, the stationary phase consists of inert particles in a dense three-dimensional matrix packed within a glass or steel column. The mobile phase can be pure water, an aqueous buffer, an organic solvent, or a mixture thereof. The stationary phase particles typically have pores and / or channels that allow only molecules smaller than a certain size to enter. Therefore, large particles are excluded from these pores and channels, and their limited interaction with the stationary phase results in them eluting as a “complete exclusion” peak at the start of the experiment. Smaller molecules (which can enter the pores) are removed from the flowing mobile phase, and the time they remain fixed in the stationary phase pores depends in part on the depth to which they penetrate into the pores. Their removal from the mobile phase flow results in longer elution times from the column and leads to separation between particles based on their size differences. A size exclusion column has a range of molecular weights that can be separated. In general, molecules larger than the upper limit are not captured by the stationary phase, molecules smaller than the lower limit will enter the solid phase completely and elute as a single band, and molecules within the range will elute at different rates, defined according to their properties, such as hydrodynamic volume. For practical examples of these methods in pharmaceutical proteins, see Bruner et al., Journal of Pharmaceutical and Biomedical Analysis. 15:1929-1935, 1997.

[0377] For example, Anicetti et al. also discussed protein purity for clinical applications (Trends in Biotechnology. 7:342-349, 1989). More recent techniques for analyzing protein purity include, but are not limited to, LabChip GXII, an automated platform for rapid analysis of proteins and nucleic acids that provides high-throughput analysis of protein titers, sizes, and purity. In some non-limiting embodiments, clinical-grade activatable preproteins can be obtained by methods such as utilizing a combination of chromatographic materials in at least two orthogonal steps (see, for example, Therapeutic Proteins: Methods and Protocols. Vol. 308, Eds., Smales and James, Humana Press Inc., 2005). Typically, protein reagents (e.g., activatable preproteins) are substantially free of endotoxins, as measured according to techniques known in the art and described herein.

[0378] This also includes protein solubility assays. For example, such assays can be used to determine optimal growth and purification conditions for recombinant production, to optimize buffer selection, and to optimize the selection of activatable preproteins and their variants. Solubility or aggregation can be assessed based on a variety of parameters, including temperature, pH, and the presence or absence of salts and other additives. Examples of solubility screening assays include, but are not limited to, microplate-based methods for measuring protein solubility using turbidity or other measurements as endpoints; high-throughput assays for analyzing the solubility of purified recombinant proteins (see, for example, Stevanall et al., Biochim Biophys Acta. 1752:6-10, 2005); monitoring and measuring in vivo protein folding and solubility using structural complementation of genetically marked proteins (see, for example, Wigley et al., Nature Biotechnology. 19:131-136, 2001); and electrochemical screening for the solubility of recombinant proteins in E. coli using scanning electrochemical microscopy (SECM) (see, for example, Nagamine et al., Biotechnology and Bioengineering. 25 96:1008-1013, 2006), etc. Activable proproteins with increased solubility (or reduced aggregation) can be identified or selected using conventional techniques in the art, including simple in vivo assays for protein solubility (see, for example, Maxwell et al., Protein Sci. 8: 1908-11, 1999).

[0379] Protein solubility and aggregation can also be measured using dynamic light scattering (DLS). Aggregation is a general term encompassing various types of interactions or properties, including soluble / insoluble, covalent / non-covalent, reversible / irreversible, and native / denatured interactions and characteristics. For protein therapeutics, the presence of aggregation is generally considered undesirable due to concerns that it may induce immunogenic reactions (e.g., small aggregates) or potentially cause adverse events upon administration (e.g., microparticles). Dynamic light scattering is a technique used to determine the size distribution spectrum of small particles in suspension or polymers such as proteins in solution. This technique (also known as photon-correlated spectroscopy (PCS) or quasi-elastic light scattering (QELS)) uses scattered light to measure the diffusion rate of protein particles. Fluctuations in scattering intensity can be observed due to the Brownian motion of molecules and particles in solution. This motion data can be routinely processed to obtain the size distribution of the sample, where the size is given by the Stokes radius or hydrodynamic radius of the protein particles. Hydrodynamic size depends on mass and shape (conformation). Dynamic scattering can detect the presence of very small amounts of aggregated proteins (<0.01% by weight), even in samples containing a wide range of masses. It can also be used to compare the stability of different formulations, including, for example, applications that rely on real-time monitoring of changes at elevated temperatures. Therefore, some embodiments include using dynamic light scattering to analyze the solubility and / or presence of aggregation in samples containing the activatable proprotein of this disclosure.

[0380] Although the foregoing embodiments have been described in detail by way of illustration and example for the purpose of clarity, it will be readily apparent to those skilled in the art, based on the teachings of this disclosure, that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims. The following embodiments are provided for illustrative purposes only and not for limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially similar results.

[0381] 【Example】

[0382] [Example 1: Engineering of “IL-2-linker-IL-2RA” and “IL-2RA-linker-IL-2” activated proproteins]

[0383] To reduce the toxicity of IL-2-related therapeutics, IL-2-linker-IL-2Rα (hereinafter referred to as ILR fusion protein) and IL-2Rα-linker-IL-2 fusion protein (hereinafter referred to as RLI fusion protein) are produced as prodrugs or activated proproteins. The prodrugs, in their activated form, exhibit very low activity. Upon cleavage by a protease-specific linker sequence designed within the prodrug, complete or near-complete activity can be restored (see, for example...). Figures 4A-4E ).

[0384] The exemplary fusion protein used human IL-2-T3 with triple mutations of V69A, Q74P, and I128T, which has a higher binding affinity for IL-2Rα. The TEV protease cleavage site was used to provide proof of concept in order to restore IL-2 activity.

[0385] Plasmids encoding single-stranded IL-2-linker-IL-2Rα (ILR) and IL-2Rα-linker-IL-2 (RLI) with or without Fc fusion were constructed using standard gene synthesis and then subcloned into the pTT5 expression vector. Figure 2A , 2B Schematic diagrams illustrating illustrative forms of ILR fusion proteins are shown in 2D and 2E. Figure 3A , 3B Schematic diagrams illustrating illustrative RLI fusion protein forms are shown in 3D and 3E.

[0386] IL-2-Stable Connector with C-End His Label - IL-2Rα Form ( Figure 2A Illustrative proteins include P1522 and P1525. The IL-2-TEV-IL-2Rα form with a C-terminal His tag ( Figure 2B Illustrative proteins include P1630, P1664, and P1667. Fc-TEV-IL-2-Stable Linker-IL-2Rα Form ( Figure 2D Illustrative proteins include P1523 and P1526. The Fc-stable linker-IL-2-TEV-IL-2Rα form ( Figure 2E Illustrative proteins of the IL-2-Stable Connector-IL-2Rα-TEV-Fc form include P1631, P1665, and P1668. Illustrative proteins of the IL-2-Stable Connector-IL-2Rα-TEV-Fc form include P1524 and P1527. Illustrative proteins of the IL-2-TEV-IL-2Rα-Stable Connector-Fc form include P1632, P1666, and P1669.

[0387] IL-2Rα stable connector with C-terminal His tag - IL-2 form ( Figure 3A Illustrative proteins include P1528 and P1531. The IL-2Rα-TEV-IL-2 form with a C-terminal His tag (…) Figure 3B Illustrative proteins include P1633, P1773, and P1776. Fc-TEV-IL-2Rα-Stable Linker-IL-2 Form ( Figure 3D Illustrative proteins include P1529 and P1532. Fc-stable linker-IL-2Rα-TEV-IL-2 form ( Figure 3EIllustrative proteins of the IL-2Rα-Stable Connector-IL-2-TEV-Fc form include P1634, P1774, and P1777. Illustrative proteins of the IL-2Rα-TEV-IL-2-Stable Connector-Fc form include P1530 ​​and P1533. Illustrative proteins of the IL-2Rα-TEV-IL-2-Stable Connector-Fc form include P1635, P1775, and P1778.

[0388] In P1522, P1523, P1524, P1528, P1529, and P1530, the linker length between IL-2 and IL-2Rα is 5 amino acids. In P1525, P1526, P1527, P1531, P1532, and P1533, the linker length is 10 amino acids. In P1630, P1631, P1632, P1633, P1634, and P1635, the linker length is 11 amino acids. In P1664, P1665, P1666, P1773, P1774, and P1775, the linker length is 15 amino acids. In P1667, P1668, P1669, P1776, P1777, and P1778, the linker length between IL-2 and IL-2Rα is 19 amino acids.

[0389] For the ILR form, a true protease cleavage site (PS) is introduced into the linker between IL-2 and IL-2Rα. The potential O-glycosylation site in IL-2 is replaced with alanine (T3A). A disulfide bond is 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).

[0390] For the Fc-stable linker-IL-2Rα-PS-IL-2 form, a true protease cleavage site (PS) is introduced into the linker between IL-2Rα and IL-2. A potential O-glycosylation site in IL-2 is replaced with an alanine residue (T3A). A disulfide bond is introduced between IL-2 and IL-2Rα. At least one cysteine ​​mutation is introduced in K35, R38, or E61 of IL-2 and D04, H120, K38, or S39 of IL-2Rα. Exemplary proteins include P1725, P1726, P1727, P1728, P1729, and P1730.

[0391] For the design of the activatable protease, the actual protease cleavage site (PS) is introduced into the linker between IL-2 and IL-2Rα to obtain the IL-2-PS-IL-2Rα-stable linker-Fc form. The potential O-glycosylation site in IL-2 is replaced with alanine (T3A). A plasmid encoding IL-2-PSs-IL-2Rα-stable linker-Fc is constructed using standard gene synthesis and subcloned into the pTT5 expression vector with protease cleavage sites flanking the linker. Exemplary proteins include P1779, P1780, P1781, P1782, P1783, P1784, and P1785. P1786 was generated as a control protein and has no cleavage site between IL-2 and IL-2Rα.

[0392] For the design of the activatable protease, different true protease cleavage sites (PS) were introduced into the linkers between Fc / IL-2 and IL-2 / IL-2Rα, respectively. Potential O-glycosylation sites in IL-2 were replaced with alanine (T3A). In one construct, potential N-glycosylation sites in IL-2R were replaced with alanine (N49A and N68A). IL-2-D10 was also tested in one construct. A plasmid encoding Fc-PS1-IL-2-PS2-IL2Rα was constructed using standard gene synthesis and subcloned into the pTT5 expression vector with linker-flanking protease cleavage sites. Exemplary proteins include P1834, P1835, P1836, P1837, P1838, P1839, P1840, P1841, P1842, P1843, P1844, P1845, P1846, P1847, P1848, P1849, and P1850.

[0393] Wild-type IL-2 and IL-2 mutant proteins with low binding affinity for IL-2Rα were also tested in the form of the Fc stable linker-IL-2-TEV-IL-2Rα. A potential O-glycosylation site was replaced by an alanine residue (T3A). IL-2 mutant proteins tested included IL-2-F42A, IL-2-Y45A, and IL-2-F42A-Y45A. Exemplary proteins include P1946, P1947, P1948, and P1949. The IL-2-E61S / IL-2Rα-K38S combination was also tested. Exemplary protein includes P1972.

[0394] The ILR form was also tested in the antibody fusion form. The ILR is fused to the C-terminus of the antibody heavy chain, which has a protease cleavage site between the heavy chain and IL-2 or between IL-2 and IL-2Rα. A potential O-glycosylation site in IL-2 is replaced with alanine (T3A), and the C-terminal lysine (K) on the heavy chain is deleted. Due to the use of IgG4-Fd, cysteine ​​217 on the heavy chain is replaced with serine. Exemplary proteins include P14501950, P14501951, P14501952, and P14501953.

[0395] Production, purification, and characterization

[0396] Fc fusion proteins were generated by transient transfection into Expi293 cells and purified using a two-step purification process involving MabSelect SuRe chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare). His-tagged proteins were also generated by transient transfection into Expi293 cells and purified using a two-step purification process involving nickel affinity chromatography (GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare).

[0397] The purified protein was characterized by SDS-PAGE for purity assessment and showed good purity, for example, Figure 6A , 6B The numbers 10A, 10B, 13A, 13B, 16A, 16B, 19A, 19B, 22A, 22B, 25A, 25B, 27A, and 27B are shown.

[0398] Purified proteins with corresponding cleavage sites were cleaved by proteases. The proteases tested were: TEV, uPA (R&D, Cat#1310-SE-010), proteolytic enzyme (R&D, Cat#3946-SEB-010), and MMP-2 (R&D, Cat#902-MP-010). P1529, P1532, P1630, P1631, and P1632 could not be cleaved by TEV, while other proteins could be cleaved by TEV, such as... Figure 6C As shown. P1664, P1665, P1666, P1667, P1668, and P1669 can be cut by TEV, as follows. Figure 10C As shown. P1719, P1721, P1722, P1723, P1724, P1725, and P1726 can be partially cleaved by uPA protease, as... Figure 13C As shown. P1773, P1774, P1775, P1776, P1777, and P1778 can be cleaved by TEV, and P1779, P1780, P1781, P1782, P1783, P1784, and P1785 can be cleaved by uPA protease, as shown. Figure 16C As shown.

[0399] like Figure 19C As shown, purified proteins can be partially or completely cleaved by uPA, proteolytic enzymes, or MMP-2. Figure 19D As shown, P1842 and P1847 can be cut by both uPA and MMP-2 simultaneously. P1946, P1947, P1948, and P1949 can be partially cut by TEV, as shown... Figure 22C As shown. P14501950, P14501951, P14501952, and P14501953 can be completely or partially cleaved by MMP-2, uPA, or proteolytic enzymes, such as... Figure 25C As shown. P1972 can be partially cut by TEV, as... Figure 27C As shown.

[0400] The purified protein was also characterized for homogeneity assessment by high-performance liquid chromatography (HPLC). 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 Among 11A-11F, 14A-14D, 17A-17D, 20A-20D, 23A-23D, 26A-26D, and 27D, most proteins show a single peak, indicating good homogeneity.

[0401] [Functional Assay - Proliferation]

[0402] The purified protein was subjected to proliferation assays before and after cleavage. M-07e (IL-2Rβ / γc) cells were cultured in RPMI 1640 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 during the logarithmic growth phase and washed twice with PBS. 90 μl of cell suspension (2 × 10⁻⁶) was used to measure the proliferation of the purified protein. 4 Cells per well were seeded into 96-well plates and incubated for 4 hours at 37°C and 5% CO2 in assay medium (RPMI 1640 supplemented with 10% FBS and 1% NEAA) for cytokine starvation. An IL-2 control and purified protein sample were prepared in assay medium to an initial concentration of 300 nM, followed by 1 / 3 serial dilutions. 10 μl of the diluted protein was added to the corresponding well and incubated at 37°C and 5% CO2 for 72 hours. Viable cell counts were measured using a Cell Counting Kit-8 (CCK-8, Dojindo, CK04). Results were displayed as... Figure 8A-8L 9A-9E, 12A-12F, 15A-15E, 18A-18N, 21A-21Q, 24A-24D and 28.

[0403] No activity was detected in fusion proteins lacking TEV cleavage sites from IL-2 stable linker-IL-2Rα forms (P1522 and P1525) and IL-2Rα stable linker-IL-2 forms (P1528 and P1531).

[0404] No activity was detected in the fusion proteins prior to TEV cleavage from the Fc-TEV-IL-2-stable adaptor-IL-2Rα forms (P1523 and P1526), ​​IL-2-stable adaptor-IL-2Rα-TEV-Fc forms (P1524 and P1527), Fc-TEV-IL-2Rα-stable adaptor-IL-2 forms (P1529 and P1532), and IL-2Rα-stable adaptor-IL-2-TEV-Fc forms (P1530 ​​and P1533). IL-2 activity in these fusion proteins did not recover after TEV cleavage.

[0405] For the forms with TEV cleavage sites between IL-2 and IL-2Rα, P1630, P1631, and P1632 showed no activity before TEV cleavage and could not be cleaved by TEV; P1635 showed no activity before or after TEV cleavage; P1633 and P1634 showed low activity before TEV cleavage, but recovered all or part of their activity after TEV cleavage.

[0406] For fusion proteins with a long cleavable linker (TEV cleavage site) between IL-2 and IL-2Rα, P1664, P1665, P1666, P1667, P1668, P1669, P1773, P1774, P1776, and P1777 exhibited very low activity before TEV cleavage and recovered all or part of their activity after TEV cleavage. P1775 and P1778 had no activity or very low activity before TEV cleavage, and their activity could not be recovered after TEV cleavage.

[0407] For fusion proteins with a true protease cleavage site in the linker between IL-2 and IL-2Rα, P1779, P1780, P1781, P1782, P1783, and P1785 showed no activity or very low activity before protease cleavage, and their activity was restored after protease cleavage. P1786, as a negative control, showed no activity before or after protease cleavage.

[0408] For ILR forms with a disulfide bond between IL-2 and IL-2Rα, P1719, P1721, P1722, P1723, and P1724 showed no activity or low activity before protease cleavage, but their activity was partially or completely restored after protease cleavage.

[0409] For the Fc-PS1-IL-2-PS2-IL2Rα form, the fusion protein shows no activity or very low activity before protease cleavage, and its activity is partially restored after protease cleavage at PS2, such as... Figure 21A-21O As shown. 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 either PS1 or PS2, and full activity was restored after double cleavage at both PS1 and PS2. For P1847 with superfactor D10, low activity was detected before protease cleavage, and full activity was restored after single cleavage at either PS1 or PS2, as well as after double cleavage at both PS1 and PS2; in fact, after double cleavage, this construct showed higher activity than wild-type IL-2.

[0410] For fusion proteins containing wild-type IL-2 or IL-2 mutant proteins (with lower binding affinity for IL-2Rα), P1946, P1947, P1948, and P1949 exhibited low activity before protease cleavage but recovered full activity after cleavage. P1947, P1948, and P1949, containing IL-2 mutant proteins, showed higher activity than P1946 before protease cleavage.

[0411] P1972 containing IL-2-E61S and IL-2Rα-K38S showed low activity before TEV cleavage and recovered full activity after cleavage.

Claims

1. An activatable preprotein homodimer comprising a first polypeptide and a second polypeptide, wherein: (a) The first polypeptide and the second polypeptide comprise a binding portion, a first linker, an IL-2 protein, a second linker, and an IL-2Rα protein in the N-to-C-terminal direction or the C-to-N-terminal direction; or (b) The first polypeptide and the second polypeptide comprise a binding portion, a first linker, an IL-2Rα protein, a second linker, and an IL-2 protein in the N-to-C-terminal direction or the C-to-N-terminal direction. in The binding portions of (a) and (b) contain the CH2CH3 domain of an immunoglobulin or a fragment thereof. The IL-2 proteins described in (a) and (b) contain at least 90% identical amino acid sequences to those selected from SEQ ID NO: 1-3 and 7-19 and have IL-2 activity. The IL-2Rα protein of (a) and (b) comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO: 4-6 and 20 and binds to the IL-2 protein. The binding portion of the first polypeptide binds to the binding portion of the second polypeptide. The IL-2 protein of the first polypeptide binds to the IL-2Rα protein of the second polypeptide, and The IL-2Rα protein of the first polypeptide binds to the IL-2 protein of the second polypeptide. The binding site of the IL-2 protein (which may be multiple) is masked; otherwise, this site binds to IL-2Rβ / γc and / or IL-2Rα / β / γc chains present on the surface of immune cells in vitro or in vivo. The second adapter is a cleavable adapter containing a protease cleavage site, and The length of the cuttable connector is at least 13 amino acids.

2. The activatable preprotein homodimer of claim 1, wherein the IL-2 protein comprises an amino acid sequence that is at least 90% identical to amino acids 21-153 of SEQ ID NO:

1.

3. The activatable preprotein homodimer of claim 2, wherein the IL-2 protein comprises a C145X (X is any amino acid) or C145S substitution as defined in SEQ ID NO:

1.

4. The activatable preprotein homodimer according to claim 1 or 2, The IL-2 protein described therein contains at least 92% of the same amino acid sequence as SEQ ID NO:

2. The IL-2 protein therein retains the S125 residue as defined in SEQ ID NO:

2.

5. The activatable preprotein homodimer according to claim 4, The IL-2 protein described therein contains at least 94% of the same amino acid sequence as SEQ ID NO:

2. The IL-2 protein therein retains the S125 residue as defined in SEQ ID NO:

2.

6. The activatable preprotein homodimer of any one of claims 1 to 5, wherein the IL-2Rα protein comprises an amino acid sequence that is at least 90% identical to the amino acid sequence selected from SEQ ID NO: 4 to 6 and 20.

7. The activatable preprotein homodimer of claim 6, wherein the IL-2Rα protein comprises at least 95% of the same amino acid sequence as SEQ ID NO:

6.

8. The activatable preprotein homodimer of any one of claims 1 to 7, wherein the binding portions of the first polypeptide and the second polypeptide in (a) and (b) comprise an antigen-binding domain of an immunoglobulin.

9. The activatable preprotein of any one of claims 1 to 8, wherein the binding portions of the first polypeptide and the second polypeptide of (a) and (b) comprise the CH1 and CL domains of an immunoglobulin.

10. The activatable preprotein homodimer of claim 8 or 9, wherein the antigen-binding domain comprises the VH and VL domains of an immunoglobulin.

11. The activatable preprotein homodimer of any one of claims 8 to 10, wherein the immunoglobulin is derived from an immunoglobulin class selected from IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM.

12. The activatable preprotein homodimer of any one of claims 1 to 11, wherein the cleavable linker is selected from SEQ ID NO: 21 to 114 and 442 to 477.

13. The preprotein homodimer of claim 12, wherein the cleavable linker comprises SEQ ID NO:

79.

14. The activatable preprotein homodimer of claim 12, wherein the protease cleavage site is cleaved by one or more proteases selected from metalloproteinases, serine proteases, cysteine ​​proteases, and aspartic proteases.

15. The activatable preprotein homodimer of any one of claims 12 to 14, wherein the protease cleavage site can be cleaved by one or more proteases selected from MMP1, MMP2, MMP3, MMP4, MMP5, MMP6, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, TEV protease, proteolytic enzyme, uPA, FAP, Legumin, PSA, kallikrein, cathepsin A, and cathepsin B.

16. The activatable preprotein homodimer according to any one of claims 1 to 15, wherein The length of the first connector is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids, and The length of the second connector is 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids.

17. The activatable preprotein homodimer of any one of claims 1 to 16, wherein the first linker in (a) and (b) is an uncut linker.

18. The activatable preprotein homodimer of claim 17, wherein the cleavable linker comprises SEQ ID NO:

79.

19. The activatable preprotein homodimer of claim 17, wherein protease cleavage of the second linker in (a) and / or (b) exposes the binding sites (multiple) of the first and / or second IL-2 proteins that bind to the IL-2Rβ / γc chains present on the surface of the immune cells in vitro or in vivo.

20. The activatable preprotein homodimer of any one of claims 1 to 19, wherein the immune cell is selected from one or more of T cells, B cells, natural killer cells, monocytes and macrophages.

21. The activatable preprotein homodimer of any one of claims 1 to 20, wherein the first polypeptide and the second polypeptide of (a) comprise the binding moiety, the first linker, the IL-2 protein, the second linker, and the IL-2Rα protein in the N-to-C-terminal direction.

22. The activatable preprotein homodimer of any one of claims 1 to 20, wherein the first polypeptide and the second polypeptide of (a) comprise the IL-2Rα protein, the second linker, the IL-2 protein, the first linker and the binding moiety in the N-to-C-terminal direction.

23. The activatable preprotein homodimer of any one of claims 1 to 20, wherein the first polypeptide and the second polypeptide of (b) comprise the binding moiety, the first linker, the IL-2Rα protein, the second linker, and the IL-2 protein in the N-to-C-terminal direction.

24. The activatable preprotein homodimer of any one of claims 1 to 20, wherein the first polypeptide and the second polypeptide of (b) comprise the IL-2 protein, the second linker, the IL-2Rα protein, the first linker and the binding moiety in the N-to-C-terminal direction.

25. The activatable preprotein homodimer of any one of claims 1 to 24, wherein the first polypeptide and the second polypeptide comprise at least 90%, at least 95%, at least 98%, or 100% identical amino acid sequences to those selected from SEQ ID NO: 208, 211, 232, 335, 341, 342, 343, and 394.

26. The activatable preprotein homodimer of claim 27, wherein the TEV protease cleavage site is replaced by a human protease cleavage site.

27. The activatable preprotein homodimer of claim 26, wherein the cleavable linker is selected from SEQ ID NO: 21-114 and 442-477.

28. The activatable preprotein homodimer of claim 27, wherein the cleavable linker is SEQ ID NO:

79.

29. The activatable preprotein homodimer according to any one of claims 1 to 28, which exhibits a homodimer morphology of more than 95% in physiological solution or under physiological conditions.

30. A recombinant nucleic acid molecule encoding an activatable preprotein homodimer as described in any one of claims 1 to 29.

31. A vector comprising the recombinant nucleic acid molecule of claim 30.

32. A host cell comprising the recombinant nucleic acid molecule of claim 30 or the vector of claim 31.

33. A method for producing an activatable preprotein, comprising: The steps of culturing the host cells of claim 32 under culture conditions suitable for expressing the activatable preprotein homodimer, and The step of isolating the activatable preprotein from the culture.

34. A pharmaceutical composition comprising the activatable preprotein homodimer of any one of claims 1 to 20, and a pharmaceutically acceptable carrier.

35. The pharmaceutical composition of claim 34, used to treat a subject’s disease and / or enhance the subject’s immune response, wherein the disease is selected from one or more of cancer, viral infections, and immune disorders.

36. The pharmaceutical composition according to claim 35, wherein the cancer is a primary or metastatic cancer, and is selected from one or more of the following: melanoma, renal cancer, pancreatic cancer, bone cancer, prostate cancer, small cell lung cancer, non-small cell lung cancer (NSCLC), mesothelioma, leukemia, multiple myeloma, lymphoma, liver cancer (hepatocellular carcinoma), sarcoma, B-cell malignancy, 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.

37. The pharmaceutical composition used according to claim 36, wherein... The melanoma in question is a metastatic melanoma; The renal cancer mentioned is renal cell carcinoma; or The leukemia referred to is lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, or relapsed acute myeloid leukemia.

38. The pharmaceutical composition according to any one of claims 35 to 37, wherein after administration, The activatable preprotein homodimer is activated by cleavage by proteases in cancer cells or cancerous tissue. The cleavage exposes the binding sites (multiple) of the first and / or second IL-2 proteins that bind to the IL-2Rβ / γc chains present on the surface of the immune cells, either in vitro or in vivo. This leads to the generation of activated proteins.

39. The pharmaceutical composition according to claim 38, wherein the activated protein binds to the IL-2Rβ / γc chain present on the surface of immune cells in vitro or in vivo via the IL-2 protein.

40. The pharmaceutical composition according to claim 39, wherein the immune cells are selected from one or more of T cells, B cells, natural killer cells, monocytes and macrophages.

41. The pharmaceutical composition according to any one of claims 38 to 40, wherein the binding between the IL-2 protein (may be multiple) and the IL-2Rα protein (may be multiple) in the activated protein masks the binding with T. reg The IL-2Rα / β / γc chains expressed above bind to the binding sites of the IL-2 protein (multiple chains), thereby preventing the activated protein from binding to T. reg The combination of.

42. The pharmaceutical composition according to claim 35, wherein the viral infection is selected from one or more of the following: human immunodeficiency virus (HIV), hepatitis A, hepatitis B, hepatitis C, hepatitis E, calicivirus-associated dysentery, rotavirus dysentery, Haemophilus influenzae type b (Hib). Haemophilus influenzae Type B pneumonia and invasive diseases, influenza, measles, mumps, rubella, parainfluenza-associated pneumonia, respiratory syncytial virus (RSV) pneumonia, severe acute respiratory syndrome (SARS), human papillomavirus, herpes simplex virus type 2 genital ulcers, dengue fever, Japanese encephalitis, tick-borne encephalitis, West Nile virus-associated diseases, 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.

43. The pharmaceutical composition according to claim 35, wherein the immune disorder is selected from one or more of type 1 diabetes, vasculitis, and immunodeficiency.

44. The pharmaceutical composition used according to any one of claims 35 to 43, wherein the pharmaceutical composition is administered to the subject by non-oral administration.

45. The pharmaceutical composition used according to claim 44, wherein the non-oral administration is intravenous administration.

46. ​​Use of the pharmaceutical composition of claim 34 in the preparation of a medicament for treating a disease of a subject and / or for enhancing the immune response of a subject, wherein the disease is selected from one or more of cancer, viral infections, and immune disorders.

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