Activatable interleukin-2 polypeptide and method of use thereof

Abstract

To provide activatable interleukin-2 polypeptides and use methods thereof.SOLUTION: The disclosure features a fusion protein that is a conditionally active variant of IL-2. In one aspect, the full-length polypeptide of the invention has reduced or minimal cytokine receptor-activating activity even though it contains a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g. a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Application No. 62 / 671,225, filed on 14 May 2018, U.S. Provisional Application No. 62 / 756,504, filed on 6 November 2018, and U.S. Provisional Application No. 62 / 756,507, filed on 6 November 2018. The teachings of the above applications in their entirety are incorporated herein by reference.

[0002] Sequence List This application includes an electronically submitted sequence listing in ASCII format, which is incorporated herein by reference in its entirety. The above ASCII copy, created on 14 May 2019, is named 105365-0021_SL.txt and has a size of 408,319 bytes. [Background technology]

[0003] The development of mature immune lymphoid cells from low-commitment precursors, their subsequent antigen-driven immune responses, and the suppression of these undesirable autoreactive responses are largely dependent on and regulated by cytokines (interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21, etc.) that utilize the receptors of the common gamma chain (γc) family (Rochman et al., 2009) and other family members such as IL-12, IL-18, and IL-23. IL-2 is essential for the development of Treg cells in the thymus and critically regulates several key aspects of mature peripheral Tregs and antigen-activated conventional T cells. Due to its potent T cell growth factor activity in vitro, IL-2 has been widely studied, partly because this activity has been seen as a powerful means of directly promoting immunity in patients with cancer and AIDS-HIV, for example, or as a target to counteract undesirable responses, such as transplant rejection and autoimmune diseases. In vitro studies using IL-2 provided strong theoretical justification for these studies, but the function of IL-2 in vivo is clearly far more complex, as first demonstrated in IL-2-deficient mice, and rapid, lethal autoimmune syndromes were observed rather than immune deficiency (Sadlack et al., 1993, 1995). Later, similar observations were made when the genes encoding IL-2Rα (Il2ra) and IL-2Rβ (Il2rb) were individually removed (Suzuki et al., 1995, Willerford et al., 1995).

[0004] This invention refers to conditionally active and / or targeted cytokines for use in the treatment of cancer and other diseases that depend on the upregulation or downregulation of the immune system. For example, the antitumor activity of several cytokines is well known and described, and some cytokines have already been used therapeutically in humans. Cytokines such as interleukin-2 (IL-2) have shown positive antitumor activity in patients with various types of tumors, such as renal metastasis, hairy cell leukemia, Kaposi's sarcoma, melanoma, and multiple myeloma. Other cytokines such as IFNβ, tumor necrosis factor (TNF)α, TNFβ, IL-1, IL-4, IL-6, IL-12, IL-15, and CSF have shown specific antitumor activity against several types of tumors, and therefore, they are subject to further research. [Overview of the Initiative] [Means for solving the problem]

[0005] This specification provides therapeutic proteins, nucleic acids encoding such proteins, and compositions and methods for using such proteins and nucleic acids for the treatment of diseases or disorders, such as proliferative disorders, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, etc.

[0006] The present invention features a fusion protein that is a conditionally active mutant of IL-2. In one embodiment, the full-length polypeptides of the present invention have reduced or minimal IL-2 receptor activating activity, even if they contain a functional cytokine polypeptide. For example, upon activation by cleavage of a linker that sequentially links a blocking moiety, such as a steric blocking polypeptide, to the active cytokine, IL-2, or its functional fragment or mutant protein, can bind to its receptor and perform signal transduction. If desired, the full-length polypeptide may also contain a blocking polypeptide moiety that provides additional advantageous properties. For example, the full-length polypeptide may also contain a blocking polypeptide moiety that extends the serum half-life and / or directs the full-length polypeptide to a desired site of IL-2 activity. Alternatively, the full-length fusion polypeptide may contain a serum half-life extending element and / or a target-directing domain separate from the blocking polypeptide moiety. Preferably, the fusion protein contains at least one element or domain that can extend the circulating blood half-life in vivo. Preferably, this element is removed enzymatically at a desired site in the body (e.g., cleavage by a protease in the tumor microenvironment), restoring the payload molecule (e.g., IL2 or IFNa) to pharmacokinetic properties substantially similar to those of the naturally occurring payload molecule. Preferably, the fusion protein is directed to a desired cell or tissue. As described herein, targeting is achieved either through the action of a blocking polypeptide moiety that also binds to the desired target, or through a targeting domain. A domain that recognizes a target antigen (e.g., a tumor-specific antigen) on a preferred target may be bound to a cytokine via a cleavable or non-cleavable linker. When bound with a non-cleavable linker, the targeting domain may be considered a retention domain, as it may further assist in retaining the cytokine within the tumor. The targeting domain does not necessarily have to be directly linked to the payload molecule, but may be directly linked to another element of the fusion protein. This is especially true when the targeting domain is bound with a cleavable linker.

[0007] In one embodiment, a fusion polypeptide is provided comprising an IL-2 polypeptide, or a functional fragment or mutant protein thereof, and a blocking moiety, such as a steric barrier domain. The blocking moiety can be fused to the IL-2 polypeptide directly or via a linker and separated from the cytokine polypeptide by cleavage (e.g., protease-mediated cleavage) of the fusion polypeptide within or near the fusion site, linker, or blocking moiety. For example, if the cytokine polypeptide is fused to the blocking moiety via a linker containing a protease cleavage site, the cytokine polypeptide can be released from the blocking moiety upon protease-mediated cleavage of the linker and bind to its receptor. The linker is designed to be cleaved at a site of desired cytokine activity, such as within the tumor microenvironment, thereby avoiding off-target cytokine activity and reducing the overall toxicity of cytokine therapy.

[0008] The blocking portion can also function as a serum half-life extender. In some embodiments, the fusion polypeptide further comprises separate serum half-life extenders. In some embodiments, the fusion polypeptide further comprises a target-directing domain. In various embodiments, the serum half-life extender is optionally a branched or multi-armed polyethylene glycol (PEG), a fragment that maintains binding affinity to full-length human serum albumin (HSA) or FcRn, an Fc fragment, or a nanobody that directly binds to FcRn or binds to human serum albumin.

[0009] In addition to serum half-life extending elements, the pharmaceutical compositions described herein preferably include at least one or more target-directed domains that bind to one or more target antigens or regions on a single target antigen. Hereinafter, the polypeptide constructs of the present invention are intended to be cleaved, for example, at protease cleavage sites within the disease-specific microenvironment of the subject or in the blood, and the target-directed domains(s) are intended to bind to target antigens on target cells. At least one target antigen is involved in and / or associated with a disease, disorder, or condition. Exemplary target antigens include those associated with proliferative disorders, neoplastic diseases, inflammatory diseases, immunodeficiencies, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host diseases, or host-versus-graft diseases.

[0010] In some embodiments, the target antigen is a cell surface molecule such as a protein, lipid, or polysaccharide. In some embodiments, the target antigen is located on tumor cells, virus-infected cells, bacterial-infected cells, damaged red blood cells, arterial plaque cells, or fibrous tissue cells.

[0011] Target antigens are sometimes expressed on the surface of affected cells or tissues, such as tumor or cancer cells. Examples of tumor target antigens include, but are not limited to, fibroblast-activating protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling molecule 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins comprising two antigen-binding domains that bind to two different target antigens known to be expressed on affected cells or tissues. Examples of antigen-binding domain pairs include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0012] In some embodiments, the target-directed polypeptide independently comprises an scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to a target antigen. In some embodiments, the target-directed polypeptide specifically binds to a cell surface molecule. In some embodiments, the target-directed polypeptide specifically binds to a tumor antigen. In some embodiments, the target-directed polypeptide specifically and independently binds to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide functions as a holding domain and is bound to a cytokine via a non-cleavable linker.

[0013] As described herein, the cytokine blocking moiety can bind to IL-2 and thereby block the activation of the IL-2 cognate receptor.

[0014] The present disclosure also relates to nucleic acids encoding conditionally active proteins as described herein, such as DNA, RNA, mRNA, etc., and vectors and host cells containing such nucleic acids.

[0015] The present disclosure also relates to pharmaceutical compositions containing conditionally active proteins, nucleic acids encoding conditionally active proteins, and vectors and host cells containing such nucleic acids. Typically, the pharmaceutical composition contains one or more physiologically acceptable carriers and / or additives.

[0016] The present disclosure also relates to a method of treatment comprising administering, to a subject in need thereof, an effective amount of any of the foregoing conditionally active proteins, nucleic acids encoding conditionally active proteins, vectors or host cells containing such nucleic acids, and pharmaceutical compositions. Typically, the subject has or is at risk of developing a proliferative disease, a neoplastic disease, an inflammatory disease, an immunodeficiency, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease or host-versus-graft disease.

[0017] The present disclosure also relates to the use of any of the foregoing conditionally active proteins, nucleic acids encoding conditionally active proteins, vectors or host cells containing such nucleic acids, and pharmaceutical compositions, for treating a subject in need thereof. Typically, the subject has or is at risk of developing a proliferative disease, a neoplastic disease, an inflammatory disease, an immunodeficiency, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease or host-versus-graft disease.

[0018] The present disclosure also relates to the use of a conditionally active protein, a nucleic acid encoding a conditionally active protein, a vector or a host cell containing such nucleic acid, for the manufacture of a medicament for treating diseases such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immunodeficiency, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft-versus-host disease or host-versus-graft disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] [Figure 1-1]This is a schematic diagram showing a cytokine or chemokine containing a blocking region that is activated by a protease. The blocking region can optionally function as a serum half-life extension domain. To the left of the arrow, the cytokine is linked to the blocking region via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumorous environment, the protease cleaves at the protease cleavage site on the linker, releasing the blocking region and allowing the cytokine to bind to its receptor. [Figure 1-2] This schematic diagram shows a cytokine or chemokine activated by a protease, where the HSA (blocking site) is directly bound to the target cytokine or chemokine, and the protease cleavage site is located between the HSA and the target cytokine or chemokine. To the left of the arrow, the cytokine is connected to the blocking site via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumorous environment, the protease cleaves at the protease cleavage site on the linker, releasing the blocking site and allowing the cytokine to bind to its receptor. [Figure 1-3] This is a schematic diagram showing a protease-activated cytokine or chemokine in which two or more HSAs (blocking sites) are directly bound to the target molecule. If desired, one or more of the HSAs can be bound to the cytokine or chemokine via a linker, such as a linker containing a protease cleavage site. The left side of the arrow shows that the cytokine is connected to the blocking site via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. The right side of the arrow shows that, in an inflammatory or tumorous environment, the protease cleaves at the protease cleavage site on the linker, releasing the blocking site and allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties to native cytokines (e.g., short half-life). [Figure 1-4] This schematic diagram shows protease-activated cytokines or chemokines, each containing two or more cytokines of the same or different types, with each being bound to a binding domain via a protease-cleavable linker. To the left of the arrow, the cytokine is linked to a blockage site via a protease-cleavable linker, thus blocking its ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumorous environment, the protease cleaves at the protease cleavage site on the linker, releasing the blockage site and allowing the cytokine to bind to its receptor. [Figure 2] This schematic diagram shows a protease-activated cytokine or chemokine, comprising a cytokine or chemokine polypeptide, a blocking moiety, and a serum half-life extension domain, linked by at least one protease-cleavable linker. To the left of the arrow, the cytokine is linked to a blocking moiety via a protease-cleavable linker, thus blocking its ability to bind to its receptor. The cytokine is also linked to a separate half-life extension element that extends its half-life in serum. To the right of the arrow, the protease cleaves at the protease-cleavage site on the linker, thus releasing the serum half-life extension element and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties to native cytokines (e.g., short half-life). [Figure 3]This schematic diagram shows a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a target-directing domain, linked by at least one protease-cleavable linker. To the left of the arrow, the cytokine is linked to the blocking moiety and target-directing domain via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. To the right of the arrow, the protease cleaves at the protease-cleavage site within the linker, releasing the target-directing domain and blocking moiety, allowing the cytokine to bind to its receptor. [Figure 4-1] This is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a target-directing domain, and a serum half-life extension domain, linked by at least one protease-cleavable linker, wherein the cytokine polypeptide and the target-directing domain are linked by a protease-cleavable linker. To the left of the arrow, it is shown that the cytokine polypeptide is linked to the target-directing domain, blocking moiety, and half-life extension element via a protease-cleavable linker(s), thus blocking its ability to bind to its receptor. To the right of the arrow, it is shown that in an inflammatory or tumorous environment, the protease cleaves at the protease-cleavage site on the linker(s), releasing the half-life extension element, target-directing domain, and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties to native cytokines (e.g., short half-life). [Figure 4-2]This is a schematic diagram showing a protease-activated cytokine or chemokine, comprising a cytokine or chemokine polypeptide, a blocking moiety, a target-directing domain, and a serum half-life extension domain, linked by at least one protease-cleavable linker. To the left of the arrow, the cytokine is linked to the target-directing domain, blocking moiety, and half-life extension element via a protease-cleavable linker(s), thus blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor environment, the protease cleaves at the protease-cleavage site on the linker(s), releasing the half-life extension element and blocking moiety, allowing the cytokine to bind to its receptor. The target-directing moiety remains bound, maintaining the cytokine within the tumor microenvironment. The cytokine here possesses similar pK properties to native cytokines (e.g., short half-life). [Figure 5] This is a schematic diagram showing the structure of the variable domain of an immunoglobulin molecule. Both the heavy and light chain variable domains of immunoglobulins contain three hypervariable loops, or complementarity-determining regions (CDRs). The three CDRs of the V domain (CDR1, CDR2, CDR3) form a cluster at one end of the beta barrel. CDRs are loops that connect the beta chains BC, C'-C'', and FG of the immunoglobulin fold, while the lower loops that connect the beta chains AB, CC', C''-D, and EF of the immunoglobulin fold, and the upper loops that connect the DE chain of the immunoglobulin fold, are non-CDR loops. [Figure 6] This is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety which is a serum albumin-binding domain (e.g., dAb), and a protease-cleavable linker. In the illustrated example, a non-CDR loop within the serum albumin-binding domain (e.g., sdAb) can form a binding site for the cytokine IL-2. In this example, the serum albumin binding site can be formed by the CDR of the serum albumin-binding domain. [Figure 7-1] Figures 7a–7h are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo® (Promega), a cell viability assay based on luminescence. Each proliferation assay was performed with or without HSA (Figures 7b, 7d, 7f, 7h). Each fusion protein contained an anti-HSA conjugate, and both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 7-2] Same as above. [Figure 8] Figures 8a–8f are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo (Promega), a cell viability assay based on luminescence. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 9-1] Figures 9a–9z are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo (Promega), a cell viability assay based on luminescence. Both the uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 10] The results of the protein cleavage assay described in Example 2 are shown. The fusion protein ACP16 was subjected to SDS-PAGE gel in both cleaved and uncleaved forms. As shown in the gel, cleavage was complete. [Figure 11-1] This is a series of graphs showing the results obtained from the HEK-Blue IL-2 reporter assay performed with IL-2 fusion protein and recombinant human IL-2 (Rec hIL-2). The analysis was performed using the reagent QUANTI-Blue (InvivoGen) based on the quantification of secreted alkaline phosphatase (SEAP) activity. [Figure 11-2] Same as above. [Figure 11-3] Same as above. [Figure 11-4] Same as above. [Figure 12-1] Figures 12a and 12b are two graphs showing the analysis of ACP16 (Figure 12a) and ACP124 (Figure 12b) in the HEKBlue IL-2 reporter assay in the presence of HSA. Circles indicate the activity of the uncleaved polypeptide, and squares indicate the activity of the cleaved polypeptide. Figure 12c is a graph showing the results of the CTLL-2 proliferation assay. CTLL2 cells (ATCC) were suspended and seeded at a concentration of 500,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA), and stimulated at 37°C and 5% CO2 for 72 hours using a dilution series of activatable hIL2. The activity of uncleaved and cleaved activatable ACP16 was tested. Cell activity of cleaved activatable hIL2, prepared by incubation with active MMP9, was evaluated using the luminescence-based cell viability assay CellTiter-Glo (Promega). Circles represent intact fusion proteins, while squares represent fusion proteins cleaved by proteases. [Figure 12-2] Same as above. [Figure 13-1]This graph shows the results of analyzing ACP16 in a tumor xenograft model. Figure 13a shows the tumor volume over time in mice treated with 4.4 μg of ACP16 (square), 17 μg of ACP16 (triangle), 70 μg of ACP16 (inverted triangle), 232 μg of ACP16 (black circle), and as control groups, 12 μg of wild-type IL-2 (dashed line, triangle) and 36 μg of wild-type IL-2 (dashed line, diamond). The vehicle alone is shown as a large white circle. The data shows that tumor volume decreases over time in a dose-dependent manner in mice treated with high concentrations of ACP16. [Figure 13-2] This graph shows the results of analyzing ACP124 in a tumor xenograft model. Figure 13b shows the tumor volume over time in mice treated with 17 μg of ACP124 (square), 70 μg of ACP124 (triangle), 230 μg of ACP124 (inverted triangle), and 700 μg of ACP124. The vehicle alone is indicated by a large white circle. [Figure 13-3] This graph shows the results of analyzing ACP16 and ACP124 in a tumor xenograft model. Figure 13c shows the tumor volume over time in mice treated with 17 μg of ACP16 (triangle), 70 μg of ACP16 (circle), 232 μg of ACP16 (black circle), and as a control, 17 μg of ACP124 (dashed line, triangle), 70 μg of ACP124 (dashed line, diamond), and 230 μg of ACP124 (dashed line, diamond). The vehicle alone is shown as an inverted black triangle. The data show that in mice treated with ACP16, tumor volume decreases over time in a dose-dependent manner, but no such decrease is observed with ACP124. [Figure 14-1] Figures 14a–14c are a series of spaghetti plots showing the activity of the fusion protein in the MC38 mouse xenograft model, corresponding to the data shown in Figure 13. Each line in the plot represents one mouse. [Figure 14-2] Same as above. [Figure 14-3] Same as above. [Figure 15] This graph shows the tumor volume over time in a mouse xenograft model illustrating tumor growth in control mice (white circles) and AP16-treated mice (squares). [Figure 16-1] Figure 16a shows a series of survival plots illustrating the time course of mice treated with cleavable fusion proteins. Data for mice treated with vehicle alone (gray line), 17 μg of ACP16 (dark line), and 1 μg of ACP124 (dashed line) are shown. [Figure 16-2] Figure 16b shows a series of survival plots illustrating the time course of mice treated with cleavable fusion proteins. Data for mice treated with vehicle alone (gray line), 70 μg of ACP16 (dark line), and 70 μg of ACP124 (dashed line) are shown. [Figure 16-3] Figure 16c shows a series of survival plots illustrating the time course of mice treated with cleavable fusion proteins. Data for mice treated with vehicle alone (gray line), 232 μg of ACP16 (dark line), and 230 μg of ACP124 (dashed line) are shown. [Figure 16-4] Figure 16d shows a series of survival plots illustrating the time course of mice treated with cleavable fusion proteins. Data for mice treated with vehicle alone (gray line), 232 μg of ACP16 (dark line), and 700 μg of ACP124 (dashed line) are shown. [Figure 17] This is a series of spaghetti plots showing the activity of fusion proteins in an MC38 mouse xenograft model. All mice received a total of four doses, with the exception of three highest doses of APC132, where lethal toxicity was detected after 1 week / 2 doses. The figures show the vehicle alone (top row), ACP16 at 17 μg, 55 μg, 70 μg, and 230 μg (all columns in the top row), ACP132 at 9 μg, 28 μg, 36 μg, and 119 μg (all columns in the middle row), and ACP21 at 13 μg, 42 μg, 54 μg, and 177 μg (all columns in the bottom row). Each line in the plot represents an individual animal. [Figure 18] This section describes the properties of the TriTac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Figure 19] This section describes the properties of the TriTac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Figure 20]This section describes the properties of the TriTac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Figure 21] This section describes the properties of the TriTac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Figure 22] This document describes the properties of the TriT ac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Figure 23] This document describes the properties of the TriT ac polypeptide, which can be used as an example of a fusion protein that can be cleaved by a protease. [Modes for carrying out the invention]

[0020] This specification discloses methods and compositions for manipulating and using components containing inducible cytokines. Cytokines are potent immune agonists, and for this reason, cytokines have come to be considered promising therapeutic agents in oncology. However, cytokines have proven to have a very narrow therapeutic range. Cytokines are thought to have a short serum half-life and be extremely potent. As a result, therapeutic administration of cytokines can lead to undesirable systemic effects and toxicity. These are exacerbated by the need to administer large amounts of cytokines to achieve the desired cytokine levels at the site where cytokine action is intended (e.g., tumor). Unfortunately, due to the biology of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in the treatment of tumors.

[0021] This specification discloses a fusion protein that overcomes the problems of toxicity and short half-life that have severely limited the clinical use of cytokines in oncology. The fusion protein contains a cytokine polypeptide having receptor agonist activity. However, in relation to the fusion protein, the cytokine receptor agonist activity is attenuated and the half-life in circulating blood is prolonged. The fusion protein contains protease cleavage sites, which are cleaved by proteases associated with the desired site of cytokine activity (e.g., tumor) and are typically concentrated or selectively present at the desired site of activity. Thus, the fusion protein preferentially (or selectively) and efficiently cleaves at the desired site of activity, substantially restricting cytokine activity to the desired site of activity, e.g., the tumor microenvironment. Protease cleavage at the desired site of activity, such as within the tumor microenvironment, releases a form of cytokine from the fusion protein that is far more active as a cytokine receptor agonist than the fusion protein itself (typically at least about 100 times more active than the fusion protein). The cytokines released upon cleavage of fusion proteins typically have short half-lives, often substantially similar to those of naturally occurring cytokines, further restricting cytokine activity to the tumor microenvironment. Although the half-life of the fusion protein is extended, the circulating fusion protein is attenuated, and the active cytokines are directed to the tumor microenvironment, dramatically reducing or eliminating toxicity. The fusion proteins described herein enable, for the first time, the delivery of an effective therapeutic dose of cytokines to treat tumors, substantially restricting cytokine activity to the tumor microenvironment and dramatically reducing or eliminating the undesirable systemic effects and toxicity of the cytokines.

[0022] Unless otherwise specified, all technical terms, notations, and other scientific terms used herein are intended to have meanings commonly understood by those skilled in the art to which the present invention relates. In some cases, terms having commonly understood meanings are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be interpreted as differing from those commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and are conventional methodologies by those skilled in the art, e.g., Sambrook et al. The procedure is generally performed using widely used molecular cloning methodologies, such as those described in al., Molecular Cloning: A Laboratory Manual 4th ed. (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures involving the use of commercially available kits and reagents, where necessary, are generally carried out according to the manufacturer's defined protocols and conditions, unless otherwise specified.

[0023] "Cytokines" is a well-known technical term referring to any of the group of immunomodulatory proteins (such as interleukins or interferons) secreted by immune system cells and acting as regulators of the immune system. Cytokine polypeptides usable in the fusion proteins disclosed herein include transforming growth factors such as TGF-α and TGF-β (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3); interferons such as interferon-α, interferon-β, interferon-γ, interferon-kappa, and interferon-omega; and IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, Examples include, but are not limited to, interleukins such as IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-21, and IL-25; tumor necrosis factors such as tumor necrosis factor alpha and lymphotoxins; chemokines (e.g., CXC-motif chemokines 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as polypeptide fragments that activate homologous receptors of cytokines (i.e., functional fragments of those mentioned above). "Chemokine" is a technical term referring to any of a family of small cytokines capable of inducing chemotaxis directed toward the vicinity of responsive cells.

[0024] It is well known that cytokines have short serum half-lives, often only a few minutes or hours. Even cytokine forms that have modified amino acid sequences intended to prolong their serum half-life but retain receptor agonist activity typically have short serum half-lives. As used herein, “short-half-life cytokines” refer to cytokines whose circulating half-life in the subject’s serum is substantially short, for example, those with a serum half-life of less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 60 minutes, less than 90 minutes, less than 120 minutes, less than 240 minutes, or less than 480 minutes. As used herein, short-half-life cytokines include cytokines whose sequences have not been modified to achieve a longer-than-normal half-life in the subject’s body, and polypeptides that have modified amino acid sequences intended to prolong their serum half-life but retain receptor agonist activity. Typically, short-half-life cytokine polypeptides, such as IL-2 polypeptides, have serum half-lives comparable to naturally occurring IL-2, for example, within 5, 4, 3, or 2 times that of naturally occurring IL-2. In the latter case, it is not intended to include the addition of heterologous protein domains, such as genuine half-life extenders, such as serum albumin.

[0025] Saltase is a peptidyltransferase that modifies proteins by recognizing and cleaving a localization signal at a carboxyl terminus embedded in or bound to the end of a target protein or peptide. Saltase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 125) (where X is any standard amino acid) between a Thr residue and a Gly residue on the target protein, causing the Thr residue to transiently bind to the Cys residue, which is the active site on the enzyme, forming an enzyme-thioacyl intermediate. To complete the peptide transfer and form a peptide-monomer complex, a biomolecule with an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, substituting saltase A and linking the two molecules.

[0026] As used herein, the term “steric barrier” refers to a polypeptide or polypeptide moiety that can be covalently bound to a cytokine polypeptide, either directly or indirectly, via other moieties such as linkers, for example, in the form of a chimeric polypeptide (fusion protein), but otherwise does not covalently bind to that cytokine polypeptide. A steric barrier can bind to a cytokine polypeptide non-covalently, for example, via electrostatic, hydrophobic, ionic, or hydrogen bonds. Typically, a steric barrier inhibits or blocks the activity of the cytokine moiety by being close to and of comparable size to the cytokine moiety. A steric barrier may also block by recruiting a larger protein-binding partner. An example of this is an antibody that binds to serum albumin, where the antibody itself may or may not be large enough to activate or block binding on its own, but albumin recruitment enables sufficient steric barriering.

[0027] Where used and described herein, “half-life extension element” is part of a chimeric polypeptide that extends the serum half-life and improves pK by, for example, altering its size (e.g., to exceed the renal filtration cutoff value), shape, hydrodynamic radius, or charge, or by altering parameters such as absorption, biodistribution, metabolism, and elimination.

[0028] As used herein, the terms “activatable,” “activate,” “inducible,” and “inducible” refer to the ability of a protein, i.e., a cytokine that is part of a fusion protein, to bind to its receptor and become active upon cleavage of additional elements from the fusion protein.

[0029] As used herein, “plasmid” or “viral vector” is a substance that includes a promoter that delivers the disclosed nucleic acid to a cell in an undegraded state and brings to the cell the expression of the nucleic acid molecule and / or polypeptide.

[0030] As used herein, the terms “peptide,” “polypeptide,” or “protein” are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein to mean the same thing as an amino acid sequence. It should be recognized herein that the term polypeptide is not used to suggest a specific size or number of amino acids that make up a molecule, and that the peptides of the present invention may contain at most a few or more amino acid residues.

[0031] When used throughout, “subjects” can refer to vertebrates, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animals. Such terms do not imply any particular age or sex. Therefore, it is intended to include adult and newborn subjects, regardless of sex.

[0032] As used herein, “patient” and “subject” may be used interchangeably and may refer to a subject having a disease or disorder (e.g., cancer). The terms patient or subject include human subjects and veterinary subjects.

[0033] As used herein, the terms “treatment,” “to treat,” or “to treat” mean a method of reducing the effects of a disease or condition or the symptoms of that disease or condition. Therefore, in the methods of disclosure, treatment may mean a reduction of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of the symptoms of an established disease or condition or the symptoms of that disease or condition. For example, a method for treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in question compared to a control. Therefore, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction between 10% and 100% compared to the natural or control level. Treatment is understood not to necessarily mean a cure or complete elimination of a disease, condition, or the symptoms of that disease or condition.

[0034] As used herein, the terms “prevent,” “prevent,” and “prevent” in relation to a disease or disorder mean an action, such as administering a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, before or approximately at the same time as the subject begins to exhibit one or more symptoms of the disease or disorder, that inhibits or delays the onset or exacerbation of one or more symptoms of the disease or disorder.

[0035] As used herein, references to “reducing,” “reducing,” or “inhibiting” include changes of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or greater, compared to a suitable control level. Such terms may include, but are not necessarily, the complete elimination of a function or property, such as agonist activity.

[0036] A “reduced cytokine receptor agonist” is a cytokine receptor agonist whose receptor agonist activity is reduced compared to the naturally occurring agonist of that cytokine receptor. A reduced cytokine agonist may have agonist activity at least approximately 10 times, at least approximately 50 times, at least approximately 100 times, at least approximately 250 times, at least approximately 500 times, at least approximately 1000 times, or less, compared to the naturally occurring agonist of that receptor. When a fusion protein containing a cytokine polypeptide described herein is described as “reduced” or having “reduced activity”, it means that the fusion protein is a reduced cytokine receptor agonist.

[0037] A "traceable fusion protein" is a fusion protein that does not have any domains removed, for example, by cleavage by a protease. Domains can be removed by cleavage by a protease or other enzymatic activity, but this does not happen when the fusion protein is "traceable".

[0038] As used herein, “part” refers to a portion of a molecule that has a distinct function within that molecule and whose function can be performed by that part in the context of another molecule. A part may be a chemical substance having a specific function, or a part of a biological molecule having a specific function. For example, a “blocking portion” within a fusion protein is a portion of the fusion protein that can block some or all of the activity of the fusion polypeptide. This may be a protein domain, such as serum albumin. Blocking may be achieved by a steric blocker or a specific blocker. A steric blocker blocks by size and position rather than by specific binding, one example being serum albumin. A specific blocker blocks by a specific interaction with the portion to be blocked. Specific blockers need to be tailored to a specific cytokine or active domain, and steric blockers can be used regardless of the payload, as long as they are large enough.

[0039] In general, the therapeutic use of cytokines is severely limited by their systemic toxicity. For example, TNF was initially discovered for its ability to induce hemorrhagic necrosis in some tumors and its in vitro cytotoxic effects on various tumor lines, but it has since been proven to possess strong pro-inflammatory activity, and if produced in excess, it can have dangerously harmful effects on the human body. Because systemic toxicity is an underlying problem associated with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies aimed at reducing the toxic effects while maintaining the therapeutic efficacy of this class of biological effectors are currently being evaluated.

[0040] IL-2 exerts both stimulative and modulatory functions in the immune system and, along with other members of the common gamma chain (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its action by binding to the IL-2 receptor (IL-2R), which consists of a trimer receptor composed of IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains, or a dimer βγ IL-2R (1, 3). Both IL-2R variants can transmit signals upon IL-2 binding. However, the trimer αβγ IL-2R has an affinity for IL-2 approximately 10 to 100 times higher than the dimer βγ IL-2R (3), suggesting that CD25 confers high-affinity binding between IL-2 and its receptor, but is not essential for signal transduction. Trimeric IL-2R is found in activated T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Tregs), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44 high memory phenotype (MP) CD8+, and natural killer (NK) cells are given high levels of dimeric βγ IL-2R, and these cells also respond vigorously to IL-2 in vitro and in vivo.

[0041] The expression of high-affinity IL-2R is important for T cells to respond to low concentrations of IL-2 that are transiently available in vivo. IL-2Rα expression is not observed in naive T cells or memory T cells, but is induced after antigen activation. IL-2Rβ is constitutively expressed by NK, NKT, and memory CD8+ T cells, but is also induced in naive T cells after antigen activation. γc is not strictly regulated and is constitutively expressed by all lymphoid cells. Once high-affinity IL-2R is induced by an antigen, IL-2R signaling increases IL-2Rα expression, partly via Stat5-dependent regulation of IL2ra transcription (Kim et al., 2001). This process represents a mechanism that maintains high-affinity IL-2R expression and sustains IL-2 signaling while an IL-2 supply remains.

[0042] IL-2 is captured by IL-2Rα via a large hydrophobic binding surface surrounded by a polar periphery, resulting in a relatively weak interaction (Kd 10⁻⁸M) with rapid on-off binding dynamics. However, the IL-2Rα-IL-2 binary complex introduces a very small conformational change to IL-2, promoting association with IL-2Rβ via a separate polar interaction between IL-2 and IL-2Rβ. The pseudohigh affinity of the IL2 / α / β trimer complex (i.e., Kd approximately 300 pM) clearly indicates that the trimer complex is more stable than when IL2 is bound to the α chain alone (Kd=10 nM) or the β chain alone (Kd=450 nM), as shown in Ciardelli's data. In any case, the IL2 / α / β trimer then recruits the γ chain to form a signaling quaternary complex, facilitated by a large complex binding site for the γ chain on the β chain bound to IL2.

[0043] In other words, the IL-2Rα-IL-2Rβ-IL-2 ternary complex subsequently recruits γc via weak interactions with IL-2 and stronger interactions with IL-2Rβ to produce a stable quaternary high-affinity IL-2R (Kd 10-11M, i.e., 10 pM). The formation of the high-affinity IL-2-IL-2R quaternary complex leads to signaling via tyrosine kinases Jak1 and Jak3, respectively, associated with IL-2Rβ and γc (Nelson and Willerford, 1998). The IL-2-IL-2R quaternary complex is rapidly internalized, with IL-2, IL-2Rβ, and γc being rapidly degraded, while IL-2Rα is recycled to the cell surface (Hemar et al., 1995, Yu and Malek, 2001). Therefore, their functional activities, which require sustained IL-2R signaling, necessitate a continuous supply of IL-2 to associate with IL-2Rα and form further IL-2-IL-2R signaling complexes.

[0044] Regulatory T cells actively suppress the activation of the immune system, preventing pathological autoreactive diseases and the resulting autoimmune diseases. The development of drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune diseases has been a subject of vigorous research, and has been largely unsuccessful until the development of the present invention, which can selectively deliver active interleukins to the site of inflammation. Regulatory T cells (Tregs) are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Tregs are central to immune system homeostasis and play a major role in maintaining tolerance to autoantigens and regulating the immune response to foreign antigens. Several autoimmune and inflammatory diseases, such as type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and graft-versus-host disease (GVHD), have been shown to have deficiencies in Treg cell count or Treg function.

[0045] As a result, there is great interest in developing therapies that increase the number and / or function of Treg cells. One treatment method for autoimmune diseases being considered is the transplantation of the patient's own exo-vivo-proliferated Treg cells (Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). This method has shown promise in the treatment of animal models of the disease and in several early-stage human clinical trials, but it requires personalized medicine using the patient's own T cells, is invasive, and is technically complex. Another method is therapy using low doses of interleukin-2 (IL-2). Treg cells characteristically express the high-affinity IL-2 receptor IL2Rαβγ, composed of the subunits IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), at a high constitutive level, and Treg cell proliferation has been shown to be IL-2 dependent (Malek, TR, et al., 2010, Immunity, 33:153-65).

[0046] Conversely, immune activation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) is approved for the treatment of certain cancers. High-dose IL-2 is used to treat patients with metastatic melanoma and metastatic renal cell carcinoma, which have long-term effects on overall survival.

[0047] Clinical trials of low-dose IL-2 therapy in patients with chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical trials investigating the efficacy of IL-2 in several other autoimmune and inflammatory diseases have been initiated. The rationale for using so-called low-dose IL-2 is to utilize the high IL-2 affinity of the trimer IL-2 receptor constitutively expressed in Tregs, while leaving other T cells that do not express the high-affinity receptor inactive. Aldesleukin (marketed as Proleukin® by Prometheus Laboratories, San Diego, CA), a recombinant form of IL-2 used in these trials, is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cell carcinoma, but its side effects are very severe, so its use is only recommended in hospitals where intensive care is available (web address: www.proleukin.com / assets / pdf / proleukin.pdf).

[0048] Because Treg cells respond to lower concentrations of IL-2 than many other immune cell types due to their expression of IL2R alpha, clinical trials of IL-2 in autoimmune diseases have used lower doses of IL-2 to target Treg cells (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these low doses have presented safety and tolerability issues, and the treatments used have employed daily subcutaneous injections in either chronic or intermittent 5-day treatment courses. Therefore, there is a need for autoimmune disease therapies that enhance the number and function of Treg cells, target them more specifically than IL-2, and are safer, more tolerable, and administered less frequently.

[0049] One proposed method to improve the therapeutic index of IL-2-based therapies for autoimmune diseases is to use IL-2 variants that are selective to Treg cells compared to other immune cells. The IL-2 receptor is expressed in a wide variety of immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern may contribute to its multifaceted effects on the immune system and high systemic toxicity. In particular, activated effector T cells, as well as lung epithelial cells, express IL2Rαβγ. However, activation of effector T cells directly contradicts the goal of suppressing and regulating the immune response, and activation of lung epithelial cells leads to known dose-limiting side effects of IL-2, such as pulmonary edema. In fact, the main side effect of high-dose IL-2 immunotherapy is vascular leakage syndrome (VLS), in which intravascular fluid accumulates in organs such as the lungs and liver, leading to subsequent pulmonary edema and hepatocyte damage. There is no treatment for VLS other than discontinuing IL-2. Low-dose IL-2 regimens are being tested in patients to avoid VLS, but the trade-off is that the treatment outcomes are not optimal.

[0050] According to the literature, VLS is thought to be caused by the release of inflammatory cytokines from IL-2-activated NK cells. However, there is strong evidence that pulmonary edema is due to the direct binding of IL-2 to pulmonary endothelial cells expressing low to moderate levels of functional αβγ IL-2R. Furthermore, pulmonary edema associated with the interaction between IL-2 and pulmonary endothelial cells was suppressed in CD25-deficient host mice by blocking binding to CD25 using an anti-CD25 monoclonal antibody (mAb) or by using a CD122-specific IL-2 / anti-IL-2 mAb (IL-2 / mAb) complex, thereby preventing VLS.

[0051] Treatment with interleukin cytokines other than IL-2 is even more limited. IL-15 exhibits similar immune cell stimulating activity to IL-2, but without the same inhibitory effect, making it a promising candidate for immunotherapy. Clinical trials of recombinant human IL-15 for the treatment of metastatic melanoma or renal cell carcinoma have shown clear changes in immune cell distribution, proliferation, and activation, suggesting potential antitumor activity (Conlon et al., 2014). IL-15 is currently undergoing clinical trials to treat various forms of cancer. However, IL-15 treatment is known to be associated with undesirable toxic effects, such as exacerbation of certain leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver damage. (Mishra) A.,et al.,Cance Cell,2012,22(5):645-55,Alpdogan O.et al.,Blood,2005,105(2):866-73,Conlon KC et al.,J Clin Oncol,2015,33(1):74-82).

[0052] The direct use of IL-2 as an agonist that binds to IL-2R and therapeutically modulates the immune response has been problematic due to its well-established therapeutic risks, such as its short serum half-life and high toxicity. These risks have also limited the development of therapeutics and the use of other cytokines. There is a need for new forms of cytokines that reduce these risks. This specification discloses IL-2 and IL-15 and other cytokines, functional fragments and mutant proteins of cytokines, and compositions and methods comprising conditionally active cytokines designed to address these risks and provide the necessary immunomodulatory therapies.

[0053] The present invention is designed to address the shortcomings of direct IL-2 therapy and therapies using other cytokines, and utilizes, for example, cytokine blocking moieties, such as steric blocking polypeptides, serum half-life extension polypeptides, target-directed polypeptides, linked polypeptides such as protease-cleavable linkers, and combinations thereof. Cytokines include interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs such as IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxins), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), chemokines (CXC-motif chemokines 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), which are extremely potent when administered to patients. As used herein, “chemokine” means a family of small cytokines capable of inducing chemotaxis directed toward the vicinity of responsive cells. Cytokines can provide potent therapeutics, but they are associated with undesirable effects that are clinically difficult to manage, thereby limiting their clinical applications. This disclosure relates to novel forms of cytokines that can be used in patients, in which undesirable effects are reduced or eliminated. In particular, this disclosure relates to pharmaceutical compositions comprising chimeric polypeptides (fusion proteins), nucleic acids encoding fusion proteins, and the aforementioned pharmaceutical formulations comprising cytokines or active fragments or mutant proteins of cytokines having reduced cytokine receptor activating activity compared to the corresponding cytokines. However, under selected conditions or in selected biological environments, chimeric polypeptides activate their homologous receptors and often have the same or higher potency as the corresponding naturally occurring cytokines. As described herein, this is typically achieved by using a cytokine blocking portion that blocks or inhibits the receptor activating function of the cytokine, its active fragment, or mutant protein, under general conditions rather than selected conditions such as those present at the desired site of cytokine activity (e.g., inflammatory site or tumor).

[0054] Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be prepared using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be prepared using recombinant DNA technology, synthetic chemistry, or a combination of these technologies, and can be expressed in a suitable expression system such as CHO cells. Chimeric polypeptides can similarly be prepared by the expression of a suitable nucleic acid, for example, using synthetic or semi-synthetic chemical technology. In some embodiments, the blocking moiety can be bound to the cytokine polypeptide via saltase-mediated binding. "Saltase" is a peptidyltransferase that modifies proteins by recognizing and cleaving a carboxyl terminology signal embedded in or bound to the end of a target protein or peptide. Saltase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 125) (where X is any standard amino acid) between a Thr residue and a Gly residue on the target protein, and the Thr residue transiently binds to the Cys residue, which is the active site on the enzyme, forming an enzyme-thioacyl intermediate. To complete the peptide transfer and form a peptide-monomer complex, a biomolecule with an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, substituting for saltase A and linking the two molecules.

[0055] To form a cytokine blocking moiety fusion protein, the cytokine polypeptide is first tagged with a polyglycine sequence at its N-terminus, or alternatively, with an LPXTG motif (SEQ ID NO: 125) at its C-terminus. A peptide is then bound to the blocking moiety or other element, each functioning as an acceptor site for the tagged polypeptide. In the case of binding to a domain holding the LPXTG acceptor peptide (SEQ ID NO: 125) bound via its N-terminus, the polypeptide is tagged with the polyglycine stretch at its N-terminus. In the case of binding to a domain holding the polyglycine peptide bound via its C-terminus, the polypeptide is tagged with the LPXTG saltase recognition sequence (SEQ ID NO: 125) at its C-terminus. Upon recognition of the polyglycine sequence and the LPXTG (SEQ ID NO: 125) sequence by saltase, a peptide bond is formed between the polymer peptide and the tagged polypeptide. The saltase reaction cleaves the glycine residue as an intermediate, which occurs at room temperature.

[0056] Various mechanisms can be used to eliminate or reduce the inhibition caused by the blocking portion. For example, a pharmaceutical composition may include an IL-2 polypeptide and a protease-cleavable linker containing a protease cleavage site located between or within the IL-2 blocking portion, along with a blocking portion, such as a steric blocking portion. When the protease cleavage site is cleaved, the blocking portion can dissociate from the cytokine, and the cytokine can then activate the cytokine receptor. The cytokine portion can also be blocked by a specific blocking portion, such as an antibody, that binds to an epitope found in the relevant cytokine.

[0057] Any suitable linker can be used. For example, the linker could be glycine-glycine, a saltase recognition motif, or a saltase recognition motif with a peptide sequence (Gly4Ser). n (Sequence number 126) or (Gly3Ser) nThis may include (SEQ ID NO: 127) (where n is 1, 2, 3, 4, or 5). Typically, the saltase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 125), where X is any amino acid. In some embodiments, the covalent bond is between a reactive lysine residue bound to the C-terminus of the cytokine polypeptide and a reactive aspartate residue bound to the N-terminus of the blocker or other domain. In other embodiments, the covalent bond is between a reactive aspartate residue bound to the N-terminus of the cytokine polypeptide and a reactive lysine residue bound to the C-terminus of the blocker or other domain.

[0058] Therefore, as detailed herein, the cytokine blocking moiety used (e.g., IL-2 blocking moiety) may be a steric barrier. As used herein, “steric barrier” refers to a polypeptide or polypeptide moiety that can be covalently bound to a cytokine polypeptide, for example, in the form of a chimeric polypeptide (fusion protein), directly or indirectly, via other moieties such as a linker, but otherwise does not covalently bind to the cytokine polypeptide. A steric barrier can be bound to a cytokine polypeptide non-covalently, for example, via electrostatic, hydrophobic, ionic, or hydrogen bonds. Typically, a steric barrier inhibits or blocks the activity of the cytokine moiety by being close to and of comparable size to the cytokine moiety. Steriinhibition of the cytokine moiety can be achieved by spatially separating the cytokine moiety from the steric barrier, for example, by enzymatically cleaving a fusion protein containing the steric barrier and the cytokine polypeptide at a site between the steric barrier and the cytokine polypeptide.

[0059] As will be further detailed herein, the blocking function may be due to a combination of additional functional components in the pharmaceutical composition, such as target-directing domains, serum half-life extension elements, and protease-cleavable linked polypeptides, or due to their presence. For example, serum half-life extension polypeptides may also be steric barriers.

[0060] Various factors ensure preferential delivery and activation of IL-2 to the desired IL-2 activity site, severely limiting systemic exposure to interleukins through blockade and / or targeted strategies preferentially linked to serum half-life extension strategies. In this serum half-life extension strategy, the blocked interleukins circulate for a long time (preferably 1-2 weeks or more), while the activated interleukins have the typical serum half-life of interleukins.

[0061] Compared to IL-2 with an extended serum half-life, intravenously administered IL-2 has a serum half-life of only about 10 minutes because it is distributed throughout the large extracellular space of the body (approximately 15L) in an average-sized adult. Subsequently, IL-2 is metabolized by the kidneys, and its half-life is approximately 2.5 hours (Smith, K. "Interleukin 2 immunotherapy". Therapeutic Immunology 240 (2001)). Other measurements show that IL-2 has a very short plasma half-life of 85 minutes after intravenous administration and 3.3 hours after subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of the present invention, the half-life extender is linked to the interleukin via a linker and cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full activity of the interleukin at the desired site and separate it from the non-cleavage half-life extender. In such embodiments, the fully active, free interleukin has very different pharmacokinetic (pK) properties, with a half-life of several hours rather than several weeks. Furthermore, exposure to the active cytokine is limited to the site of desired cytokine activity (e.g., inflammation site or tumor), reducing systemic exposure to the active cytokine, as well as associated toxicity and side effects.

[0062] Other cytokines envisioned in the present invention have pharmacological properties similar to IL-2 (e.g., IL-15 as reported in Blood 2011 117:4787-4795;doi:doi.org / 10.1182 / blood-2010-10-311456), and therefore, the design of the present invention addresses the drawbacks of using these agents directly and provides chimeric polypeptides that have a longer half-life and / or can be directed to a desired site of activity (e.g., an inflammatory site or a tumor).

[0063] If desired, IL-2 can be manipulated to selectively activate, for example, Treg or Teff, by either binding generally to the IL-2R complex or specifically binding to one of the three IL-2R subunits with an affinity different from that of the corresponding wild-type IL-2. For example, IL-2 polypeptides that are said to have higher affinity for the trimer form of the IL-2 receptor compared to the dimeric beta / gamma form of the IL-2 receptor compared to wild-type IL-2 include the set of mutations for SEQ ID NO: 1 (a mature IL-2 protein containing amino acids 21-153 of human IL-2 with Uniprot accession number P60568-1), namely (a) K64R, V69A, and Q74P, (b) V69A, Q74P, and T101A, (c) V69A, Q74P, and I128T, (d) N30D, V69A, Q74P, and F103S, (e) K49E, V69A, A7 3V, and K76E, (f)V69A, Q74P, T101A, and T133N, (g)N30S, V69A, Q74P, and I128A, (h)V69A, Q74P, N88D, and S99P, (i)N30S, V69A, Q74P, and I128T, (j)K9T, Q11R, K35R, V6 9A, and Q74P, (k)A1T, M46L, K49R, E61D, V69A, and H79R, (l)K48E, E68D, N71T, N90H, F103S, and I114V, (m)S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A, (n)E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T, (o)N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H, (p)N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T, (q)N26D, N29S, N30S, K54R, E67G, It may have an amino acid sequence containing one of the following: V69A, Q74P, and I92T, (r)K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T, and (s)N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V.This method can also be applied to prepare mutant proteins of other cytokines, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs such as IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxins), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), and granulocyte-macrophage colony-stimulating factor (GM-CS). For example, mutant proteins with a desired binding affinity to a congener receptor can be prepared.

[0064] As described above, any of the mutant IL-2 polypeptides disclosed herein may include, be limited to, the described sequences, or otherwise be identical to SEQ ID NO: 1. Furthermore, any of the mutant IL-2 polypeptides disclosed herein may optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine), and / or may optionally include a deletion of the alanine residue at position 1 of SEQ ID NO: 1.

[0065] Another way to improve the therapeutic index of IL-2-based therapies is to optimize the pharmacokinetics of the molecule to maximize Treg cell activation. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro requires at least 5–6 hours of exposure to effective concentrations of IL-2 (Cantrell, DA, et al., 1984, Science, 224:1312–1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes with intravenous administration and 3.3 hours with subcutaneous administration (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5–10). Due to its short half-life, maintaining circulating IL-2 levels above the level required to stimulate T cell proliferation for the necessary duration requires either high doses that result in IL-2 peak levels significantly above the EC50 of Treg cells, or frequent administration. These high IL-2 peak levels can activate the IL2Rβγ receptor, potentially leading to other unintended or adverse effects, such as VLS as described above. IL-2 analogs, or multifunctional proteins with a longer circulating half-life than IL-2, in which IL-2 is bound to a domain enabling binding to the FcRn receptor, can achieve target drug concentrations at lower doses and lower peak levels than IL-2 for a specified period. Therefore, such IL-2 analogs require lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of IL-2 drugs is also more tolerable for patients. Therapies with these characteristics clinically lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance. Alternatively, IL-2 or mutant IL-2 proteins (hereinafter, "IL-2*") can be selectively directed to the intended site of action (e.g., the site of inflammation). This targeting can be achieved by one of several strategies, such as adding a domain to the administered drug containing a blocker of the cleaved IL-2 (or mutant protein), or by targeting the domain, or by a combination of both.

[0066] In some embodiments, IL-2* partial agonists can be engineered to bind with higher or lower affinity depending on the desired target; for example, IL-2* can be engineered to bind with enhanced affinity to one of the receptor subunits and not to the others. Unlike full agonists or full antagonists, these types of partial agonists offer the ability to tune signaling properties to an amplitude that induces desired functional properties without meeting the threshold for undesirable properties. Given the specific activity of partial agonists, the IL-2 mutant repertoire can be engineered to exhibit even finer degrees of characteristic signaling activity, ranging from near-full to partial agonism to full antagonistism.

[0067] In some embodiments, IL-2* has a modified affinity for IL-2Rα. In some embodiments, IL-2* has a higher affinity for IL-2Rα than wild-type IL-2. In other embodiments, IL-2* has a modified affinity for IL-2Rβ. In one embodiment, IL-2* has enhanced binding affinity to IL-2Rβ, for example, to the N-terminus of IL-2Rβ, thereby eliminating the functional requirement for IL-2Rα. In another embodiment, IL-2* is fabricated to have increased binding affinity to IL-2Rβ but decreased binding to IL-2Rγ, resulting in defects in IL-2Rβγ heterodimerization and signal transduction.

[0068] The blocking moieties detailed below can also be used to promote binding to or activation of one or more receptors. In one embodiment, the blocking moiety is added so as to block the binding or activation of IL-2Rβγ but not alter the binding or activation of IL-2Rα. In another embodiment, the blocking moiety is added so as to reduce the binding or activation of IL-2Rα. In yet another embodiment, the blocking moiety is added so as to inhibit the binding and / or activation of all three receptors. This blockage can be mitigated by removal of the blocking moiety in a particular environment, for example, by proteolytic cleavage of the linker that links one or more blocking moieties to cytokines.

[0069] Similar methods can be applied to improve the use of other cytokines, for example, for cancer treatment, particularly as immunostimulants. For example, in this embodiment, the pharmacokinetics and / or pharmacodynamics of cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFN-alpha, IFN-beta and IFN-gamma, TNF-alpha, lymphotoxin, TGF-beta-1, TGF-beta-2, TGF-beta-3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21) can be regulated to maximize the activation of effector cells (e.g., effect T cells, NK cells) and / or cytotoxic immune response-promoting cells at a desired site of activity, such as within a tumor, preferably not systemically, but rather (e.g., to induce dendritic cell maturation).

[0070] Accordingly, the Specified Provisions provide a pharmaceutical composition comprising at least one cytokine polypeptide, for example, interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs such as IFN-alpha, IFN-beta, and IFN-gamma), tumor necrosis factors (e.g., TNF-alpha, lymphotoxins), transforming growth factors (e.g., TGF-beta-1, TGF-beta-2, TGF-beta-3), chemokines (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), or any functional fragment or mutant protein of any of the above. The polypeptide typically also comprises at least one linker amino acid sequence, which, in certain embodiments, can be cleaved by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence including HSSKLQ (SEQ ID NO: 25), GPLVGRG (SEQ ID NO: 128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130), or SGESPAYYTA (SEQ ID NO: 131). In other embodiments, the chimeric polypeptide further contains a blocking moiety, such as a sterically barred polypeptide moiety, which can block the activity of the interleukin polypeptide. The blocking moiety may include, for example, a human serum albumin (HSA) binding domain or optionally a branched or multi-armed polyethylene glycol (PEG). Alternatively, the pharmaceutical composition comprises a first cytokine polypeptide or a fragment thereof, and a blocking moiety, such as a sterically barred polypeptide moiety, wherein the blocking moiety blocks the activity of the cytokine polypeptide on the cytokine receptor, and in certain embodiments, the blocking moiety includes a protease-cleavable domain. In some embodiments, blocking and reduction of cytokine activity is easily achieved by attaching an additional domain to the N-terminus or C-terminus of the interleukin domain using a very short linker. In such embodiments, it is expected that the blockage will be mitigated by the digestion of the blocking portion, or the short linker that anchors the blocking substance to the interleukin, by the protease.Once the domain is excised or released, it becomes impossible to block cytokine activity.

[0071] A pharmaceutical composition, such as a chimeric polypeptide, may contain two or more cytokines, which may be identical or different cytokine polypeptides. For example, two or more different types of cytokines may have complementary functions. In some examples, the first cytokine is IL-2 and the second cytokine is IL-12. In some embodiments, each of the two or more different types of cytokine polypeptides may have an activity that modulates the activity of the other cytokine polypeptide. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine polypeptide is T cell activating and the second cytokine polypeptide is non-T cell activating. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine is a chemoattractant, such as CXCL10, and the second cytokine is an immune cell activator.

[0072] Preferably, the cytokine polypeptides (including functional fragments) contained in the fusion proteins disclosed herein are not mutated or manipulated to alter the properties of naturally occurring cytokines, such as receptor binding affinity and specificity or serum half-life. However, changes in the amino acid sequence from naturally occurring (including wild-type) cytokines are permissible, for example, to facilitate cloning and to achieve desired expression levels.

[0073] CD25 binding In modified IL-2 constructs, CD25 binding is often inhibited. In contrast, the IL-2 polypeptides described herein are preferably not modified to avoid CD25 binding. Preferably, the IL-2 polypeptides described herein bind to CD25. Typically, the IL-2 fusion proteins described herein are capable of CD25 binding, and the blockade is directed towards interactions with IL-2R beta and gamma (CD122 and CD132).

[0074] Blocking section The blocking moiety may be any portion that inhibits the cytokine's ability to bind to and / or activate its receptor. The blocking moiety can inhibit the cytokine's ability to bind to and / or activate its receptor by sterically blocking and / or by covalently binding to the cytokine. Examples of suitable blocking moieties include the full-length or cytokine-binding fragment or mutant protein of the cytokine's homologous receptor. Antibodies and their fragments can also be used to bind to cytokines, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies (e.g., variable domains of the heavy-chain variable domain (VH), light-chain variable domain (VL), and camel-type nanobody (VHH)), dAbs, etc. Other suitable antigen-binding domains that bind to cytokines may also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticarin, affin, affibody molecules, affimers, affitins, alphabodies, avimers, DARPin, fynomers, Knitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of cytokines to their homologous receptors. Advantageously, such portions also function as half-life extenders. For example, peptides modified by binding to water-soluble polymers such as PEG can sterically inhibit or prevent the binding of cytokines to their receptors. Polypeptides or fragments thereof with long serum half-lives may also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, and fragments and mutant proteins of such polypeptides. For example, antibodies and antigen-binding domains that bind to long-half-life proteins in serum, such as HSA, immunoglobulins, or transferrin, or to receptors that are recycled on the plasma membrane, such as FcRn or transferrin receptors, can also inhibit cytokines, especially when bound to those antigens.Examples of such antigen-binding polypeptides include single-chain variable fragments (scFv), single-domain antibodies (such as variable domains of the heavy chain variable domain (VH), light chain variable domain (VL), and camel-type nanobody (VHH)), and dAbs. Other suitable antigen-binding domains that bind to cytokines may also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, fynomers, Knitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4.

[0075] In exemplary cases, if IL-2 is a cytokine within a chimeric polypeptide, the blocking portion may be the full-length, fragment, or mutant protein of the alpha (IL-2Rα) or beta (IL-2Rβ) or gamma (IL-2Rγ) chain of the IL-2 receptor, an anti-IL-2 single-domain antibody (dAb) or scFv, Fab, an anti-CD25 antibody or its fragment, and an anti-HAS dAb or scFv, etc.

[0076] Further aspects of the present invention 1. A fusion protein comprising a cytokine moiety functionally linked to a binding moiety comprising a non-CDR loop and a cleavable linker, wherein the binding moiety can mask the binding of the cytokine to its receptor and / or the activation of the receptor by the cytokine.

[0077] 2. The fusion protein according to embodiment 1, wherein the binding portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered bulk serum protein.

[0078] 3. The fusion protein according to embodiment 1 or 2, wherein the manipulated scaffold comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold, DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffold, protein G-associated albumin-binding module, or DNA or RNA aptamer scaffold.

[0079] 4. The fusion protein according to any one of embodiments 1 to 2, wherein the binding portion can bind to bulk serum protein.

[0080] 5. The fusion protein according to any one of embodiments 1 to 3, wherein the non-CDR loop is derived from a variable domain, a constant domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof.

[0081] 6. The fusion protein according to any one of embodiments 1 to 4, wherein the binding portion further includes a complementarity-determining region (CDR).

[0082] 7. The fusion protein according to embodiment 5, wherein the binding portion can bind to the bulk serum protein.

[0083] 8. The fusion protein according to embodiment 6, wherein the bulk serum protein is a half-life extension protein.

[0084] 9. The fusion protein according to embodiment 6 or 7, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0085] 10. The fusion protein according to any one of embodiments 5 to 8, wherein the CDR loop provides a specific binding site for the bulk serum protein or the immunoglobulin light chain, or any combination thereof.

[0086] 11. The fusion protein according to any one of embodiments 1 to 9, wherein the cleavable linker includes a cleavage site.

[0087] 12. The fusion protein according to embodiment 10, wherein the cleavage site is recognized by a protease.

[0088] 13. The fusion protein according to embodiment 11, wherein the binding portion is bound to the cytokine.

[0089] 14. The fusion protein according to embodiment 11 or 11, wherein the binding portion is covalently linked to the cytokine.

[0090] 15. The fusion protein according to embodiment 11, 11, or 14, wherein the binding portion can mask the binding of the cytokine to its target through a specific intermolecular interaction between the binding portion and the cytokine.

[0091] 16. The fusion protein according to any one of embodiments 11 to 14, wherein the non-CDR loop provides a specific binding site for the binding of the portion to the cytokine.

[0092] 17. The fusion protein according to any one of embodiments 11 to 15, wherein, upon cleavage of the cleavable linker, the binding portion is separated from the cytokine, and the cytokine binds to its target.

[0093] 18. The fusion protein according to any one of embodiments 1 to 16, wherein the cytokine binds to a cytokine receptor.

[0094] 19. The fusion protein according to embodiment 17, wherein the cytokine receptor includes a type I cytokine receptor, a type I IL receptor, a type II IL receptor, a chemokine receptor, or a tumor necrosis receptor superfamily receptor.

[0095] 20. The fusion protein according to any one of embodiments 1 to 18, wherein the cleavable linker includes a cleavage site.

[0096] 21. The fusion protein according to embodiment 20, wherein the cleavage site is recognized by a protease.

[0097] 22. The fusion protein according to embodiment 21, wherein the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloprotease, gelatinase, or asparagine peptidylase.

[0098] 23. The protease cleavage sites are: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, and Cutinidine, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, prasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, M The fusion protein according to embodiment 21, which is recognized by MP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

[0099] 24. A conditionally active binding protein comprising a non-CDR loop, a cytokine, and a binding portion (M) comprising a cleavable linker (L), wherein the non-CDR loop can bind to the cytokine, and the binding portion can inhibit the binding of the cytokine to its receptor and / or inhibit the activation of the receptor by the cytokine.

[0100] 25. The binding portion is a conditionally active binding protein according to embodiment 24, which can bind to a half-life extension protein.

[0101] 26. The conditionally active binding protein according to embodiment 24 or 25, wherein the binding portion is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein.

[0102] 27. The manipulated scaffold comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold, DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffold, protein G-associated albumin-binding module, or DNA or RNA aptamer scaffold, the conditionally active binding protein as described in Embodiment 26.

[0103] 28. The non-CDR loop is derived from a variable domain, a constant domain, a C1 set domain, a C2 set domain, an I domain, or any combination thereof, and is a conditionally active binding protein according to any one of embodiments 24 to 27.

[0104] 29. The binding portion further comprises a complementarity-determining region (CDR), wherein the conditionally active binding protein is according to any one of embodiments 24 to 28.

[0105] 30. The binding portion includes a binding site specific to bulk serum protein, wherein the binding portion is a conditionally active binding protein according to any one of embodiments 24 to 29.

[0106] 31. The conditionally active binding protein according to embodiment 30, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0107] 32. A conditionally active binding protein according to any one of embodiments 29 to 31, wherein the CDR provides a specific binding site for the bulk serum protein or the immunoglobulin light chain, or any combination thereof.

[0108] 33. A conditionally active binding protein according to any one of embodiments 29 to 32, wherein the binding portion can mask the binding of the cytokine to its target through a specific intermolecular interaction between the binding portion and the cytokine.

[0109] 34. A conditionally active binding protein according to any one of embodiments 29 to 33, wherein the non-CDR loop provides a specific binding site for the binding portion to the cytokine.

[0110] 35. The cytokine is a conditionally active binding protein according to any one of embodiments 24 to 34, which binds to a cytokine receptor.

[0111] 36. The cytokine receptor is a conditionally active binding protein according to embodiment 35, comprising a type I cytokine receptor, a type I IL receptor, a type II IL receptor, a chemokine receptor, or a tumor necrosis receptor superfamily receptor.

[0112] 37. The cleavable linker includes a cleavage site, and the conditionally active binding protein is as described in embodiments 24 to 36.

[0113] 38. The cleavage site is recognized by a protease, the conditionally active binding protein according to embodiment 37.

[0114] 39. The conditionally active binding protein according to embodiment 38, wherein the protease cleavage site is recognized by serine protease, cysteine ​​protease, aspartate protease, threonine protease, glutamate protease, metalloprotease, gelatinase, or asparagine peptide lyase.

[0115] 40. The protease cleavage sites are: cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hK1, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actin Nidaine, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, prasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase A conditionally active binding protein according to embodiment 38, recognized by -ase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

[0116] 41. A conditionally active binding protein according to embodiment 24, further comprising a half-life extension domain bound to the binding portion, wherein the half-life extension domain provides a safety switch to the binding protein, and upon cleavage of the linker, the binding protein is activated by the separation of the binding portion and the half-life extension domain from the cytokine, thereby separating the binding protein from the safety switch.

[0117] 42. The conditionally active binding protein according to embodiment 41, wherein the linker cleavage occurs in the tumor microenvironment.

[0118] 43. A conditionally active binding protein comprising a binding portion that binds to a cytokine via a non-CDR loop within the binding portion, wherein the binding portion is further linked to a half-life extension domain and comprises a cleavable linker, the binding protein having an extended half-life prior to its activation by cleavage of the linker, the binding portion and the half-life extension domain being separated from the cytokine upon activation, and the binding protein having no extended half-life in its activated state.

[0119] 44. The conditionally active binding protein according to embodiment 43, wherein the linker cleavage occurs in the tumor microenvironment.

[0120] In vivo half-life extension factor Preferably, the chimeric polypeptide contains an in vivo half-life extender. Increasing the in vivo half-life of therapeutic molecules that have short half-lives in nature allows for more acceptable and manageable dosing regimens without sacrificing efficacy. As used herein, “half-life extender” is part of a chimeric polypeptide that increases the in vivo half-life and improves pK by, for example, altering its size (e.g., to exceed the renal filtration cutoff value), shape, hydrodynamic radius, charge, or by altering parameters such as absorption, biodistribution, metabolism, and elimination. Exemplary methods of improving the pK of a polypeptide involve the expression of elements of the polypeptide chain that bind to receptors that are recycled to the cell plasma membrane rather than being degraded by lysosomes, such as FcRn receptors and transferrin receptors on endothelial cells. Three proteins, such as human IgG, HSA (or fragments), and transferrin, survive in human serum much longer than would be predicted from their size alone, and this is due to their binding ability to receptors that are recycled rather than degraded by lysosomes. These proteins, or their fragments that retain FcRn binding, are routinely ligated to other polypeptides to extend their serum half-lives. In one embodiment, the half-life extender is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may also be ligated directly to the pharmaceutical composition or via a short linker. Fragments of HSA may also be used. HSA and its fragments can function as both blocking moieties and half-life extenders. Human IgG and Fc fragments can also perform similar functions.

[0121] The serum half-life extension elements may also be antigen-binding polypeptides that bind to long-lived serum proteins such as serum albumin and transferrin. Examples of such polypeptides include antibodies and their fragments, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies (e.g., variable domains of the heavy chain variable domain (VH), light chain variable domain (VL), and camel-type nanobody (VHH)), and dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, fynomers, Knitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0122] Some preferred serum half-life extenders are polypeptides containing a complementarity-determining region (CDR) and, optionally, a non-CDR loop. Advantageously, such serum half-life extenders can extend the serum half-life of cytokines and also function as cytokine inhibitors (e.g., via steric barriers, non-covalent interactions, or a combination thereof) and / or as target-directed domains. In some cases, the serum half-life extender is a domain derived from an immunoglobulin molecule (Ig molecule) or an engineered protein scaffold that mimics the structure and / or binding activity of an antibody. Ig may be any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of the Ig molecule folds into a series of parallel beta chains linked by loops. In the variable region, three of the loops constitute the "complementarity-determining region" (CDR), which determines the antigen-binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2, and CH3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which are hypervariable in sequence and / or involved in antigen recognition and / or usually form structurally defined loops, and are interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxyl terminus. In some embodiments of this disclosure, at least some or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the “non-CDR loop” of the binding site described herein. As shown in Figure 5, the variable domain of the immunoglobulin molecule has several beta chains arranged in two sheets.The variable domains of both the heavy and light chains of immunoglobulins contain three hypervariable loops, or complementarity-determining regions (CDRs). The three CDRs of the V domain (CDR1, CDR2, CDR3) form a cluster at one end of the beta barrel. The CDRs are loops connecting the beta chains BC, C'-C'', and FG of the immunoglobulin fold, while the lower loops connecting the beta chains AB, CC', C''-D, and EF of the immunoglobulin fold, as well as the upper loops connecting the DE chain of the immunoglobulin fold, are non-CDR loops. In some embodiments of this disclosure, at least several amino acid residues of the constant domain, CH1, CH2, or CH3, are part of the “non-CDR loops” of the binding moiety described herein. In some embodiments, the non-CDR loops include one or more of the following: AB, CD, EF, and DE loops of the C1 set domain of Ig or an Ig-like molecule; AB, CC', EF, FG, BC, and EC' loops of the C2 set domain of Ig or an Ig-like molecule; and DE, BD, GF, A(A1A2)B, and EF loops of the I(intermediate) set domain of Ig or an Ig-like molecule.

[0123] Within the variable domain, the CDR is thought to be involved in antigen recognition and binding, and the FR residues are considered a scaffold for the CDR. However, in certain cases, some FR residues play a crucial role in antigen recognition and binding. The residues in the framework region that influence Ag binding can be divided into two categories. The first are FR residues that come into contact with the antigen and are therefore part of the binding site, and some of these residues are adjacent to the CDR. Other residues are adjacent to the CDR but are close in the 3-D structure of the molecule, e.g., in the heavy chain loop. A serum half-life extension domain (e.g., a domain containing the CDR) may contain at least one non-CDR loop. In some embodiments, the non-CDR loop provides a binding site for binding to cytokines, bulk serum proteins, or other target antigens.

[0124] The serum half-life extension element includes, in addition to or alternatively to, a CDR loop. In some embodiments, the non-CDR loop is modified to generate an antigen-binding site specific to a desired target antigen, such as a bulk serum protein like albumin, or to a cytokine moiety or other target antigen. Various techniques can be used to modify the non-CDR loop, such as site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid exogenous to the non-CDR loop amino acid sequence, amino acid substitution, etc. In some examples, an antigen peptide is inserted into the non-CDR loop. In some examples, the non-CDR loop is replaced with an antigen peptide. Modification to generate an antigen-binding site may, in some cases, be performed on only one non-CDR loop. In other cases, two or more non-CDR loops are modified. For example, the modification is in one of the non-CDR loops shown in Figure 5, namely AB, CC', C”D, EF, and DE. In some cases, the modification is within the DE loop. In other cases, the modification is in all four loops: AB, CC', C”-D, and EF.

[0125] In some examples, the serum half-life extender has double-binding specificity and contains a CDR that specifically binds to bulk serum proteins such as serum albumin, and a non-CDR loop that specifically binds to and blocks the cytokine domain. In other examples, the serum half-life extender contains a CDR that specifically binds to a target antigen such as a cytokine domain or other target antigen, and a non-CDR loop that specifically binds to bulk serum proteins such as serum albumin. Preferably, the serum half-life extender inhibits the binding of the cytokine domain to a homologous cytokine receptor, for example, via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0126] In some embodiments, serum half-life extenders directly bind to cytokines via non-covalent bonds and inhibit their activity.

[0127] In certain cases, the binding site binds to cytokines via one or more of the AB, CC', C”D, and EF loops, and to bulk serum proteins such as albumin via one or more of the BC, C'C”, and FG loops. In certain cases, the binding site binds to bulk serum proteins such as albumin via its AB, CC', C”D, or EF loop, and to cytokines via its BC, C'C”, or FG loop. In certain cases, the binding site binds to bulk serum proteins such as albumin via its AB, CC', C”D, and EF loops, and to cytokines via its BC, C'C”, and FG loops. In certain cases, the binding site binds to bulk serum proteins such as albumin via one or more of the AB, CC', C”D, and EF loops, and to cytokines via one or more of the BC, C'C”, and FG loops.

[0128] The binding site is any type of polypeptide. For example, in some cases, the binding site is a native peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding site is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolds (as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine knot peptide, lipocalin, 3-helix bundle scaffolds, protein G-associated albumin-binding modules, or DNA or RNA aptamer scaffolds.

[0129] In some cases, the serum half-life extension element binds to a cytokine domain via its non-CDR loop, and the cytokine domain is further connected to a target-directed domain as described herein. In some cases, the serum half-life extension element includes a binding site to bulk serum proteins. In some embodiments, the CDR provides a binding site to bulk serum proteins. Bulk serum proteins are, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDR forms a binding site to immunoglobulin light chains, such as Igκ free light chains or Igλ free light chains.

[0130] An example of a conditionally active protein is shown in Figure 6. In the illustrated example, a non-CDR loop within the serum albumin-binding domain (e.g., dAb) can form a binding site for the cytokine IL-2. In this example, the serum albumin binding site can be formed by the CDR of the serum albumin-binding domain.

[0131] The serum half-life extension component can be any type of binding domain, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding site is a single-chain variable fragment (scFv), a single-domain antibody, e.g., a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a camel-derived nanobody variable domain (VHH). In other embodiments, the binding site is a non-Ig binding domain, i.e., antibody mimics such as antikalin, affin, affibody molecules, affimers, afitins, alpha bodies, avimers, DARPin, fynomers, Knitz domain peptides, and monobodies.

[0132] In other embodiments, the serum half-life extender may be a water-soluble polymer or a peptide bound to a water-soluble polymer such as PEG. As used herein, “PEG,” “polyethylene glycol,” and “poly(ethylene glycol)” are synonymous and encompass any non-peptide water-soluble poly(ethylene oxide). The term “PEG” also means a polymer containing a majority, i.e., more than 50%, of the -OCH2CH2- repeating subunit. With respect to a particular form, PEG can take any number of different molecular weights, as well as structural or geometric forms, e.g., “branched,” “linear,” “fork-type,” “polyfunctional,” etc., details of which are described below. PEG is not limited to a specific structure and can be linear (e.g., encapsulated ends, e.g., alkoxyPEG or bifunctional PEG), branched or multi-armed (e.g., fork-type PEG or PEG bound to a polyol core), or dendritic (or star-shaped) structure, each with or without one or more degradable bonds. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating trippolymers, random trippolymers, and block trippolymers. PEG can be conjugated to polypeptides and peptides by any suitable method. Typically, a reactive PEG derivative such as N-hydroxysuccinamidyl ester PEG is reacted with a peptide or polypeptide containing amino acids having side chains containing amine, sulfhydryl, carboxylic acid, or hydroxyl functional groups, such as cysteine, lysine, asparagine, glutamine, theonine, tyrosine, serine, aspartic acid, and glutamic acid. Target-directed domains and retention domains

[0133] For certain applications, it may be desirable to maximize the time a constituent remains in its desired location within the body. This can be achieved by including an additional domain in the chimeric polypeptide (fusion protein) to influence its movement within the body. For example, a chimeric nucleic acid may encode a domain that directs the polypeptide to a site within the body, such as a tumor cell or an inflammatory site (this domain is called a “target-directing domain”), and / or a domain that retains the polypeptide at that site within the body, such as a tumor cell or an inflammatory site (this domain is called a “retention domain”). In some embodiments, the domain can function as both a target-directing domain and a retention domain. In some embodiments, the target-directing domain and / or retention domain are specific to a protease-rich environment. In some embodiments, the encoded target-directing domain and / or retention domain are specific to regulatory T cells (Tregs) and target, for example, the CCR4 receptor or the CD39 receptor. Other suitable target-directed and / or retaining domains include those having a congeneral ligand overexpressed in inflammatory tissue, e.g., a congeneral ligand of the IL-1 receptor or the IL-6 receptor. In other embodiments, suitable target-directed and / or retaining domains include those having a congeneral ligand overexpressed in tumor tissue, e.g., a congeneral ligand of Epcam, CEA, or mesothelin. In some embodiments, the target-directed domain is linked to the interleukin via a linker and cleaved at the site of action (e.g., by an inflammation or cancer-specific protease) to release the full activity of the interleukin at the desired site. In some embodiments, the target-directed and / or retaining domain is linked to the interleukin via a linker and remains at the desired site without being cleaved at the site of action (e.g., by an inflammation or cancer-specific protease).

[0134] The antigens selected are, in some cases, expressed on the surface of affected cells or tissues, such as tumor or cancer cells. Antigens useful for targeting and retention to tumors include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins comprising two targeting domains and / or retention domains that bind to two different target antigens known to be expressed on affected cells or tissues. Exemplary pairs of antigen-binding domains include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0135] Suitable target-directed and / or retention domains include antigen-binding domains, such as antibodies and their fragments, including polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibody single-chain variable fragments (scFv), single-domain antibodies (such as variable domains of the heavy chain variable domain (VH), light chain variable domain (VL), and camel-type nanobody (VHH)), and dAbs. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as antikalin, affilin, affibody molecules, affimers, affitins, alpha bodies, avimers, DARPin, fynomers, Knitz domain peptides, monobodies, and binding domains based on other manipulated scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding moieties of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0136] In some embodiments, the target-directing domain and / or retention domain specifically bind to cell surface molecules. In some embodiments, the target-directing domain and / or retention domain specifically bind to tumor antigens. In some embodiments, the target-directing polypeptide specifically and independently binds to tumor antigens selected from at least one of the following: fibroblast-activating protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling molecule 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide specifically and independently binds to two different antigens, at least one of which is a tumor antigen selected from fibroblast-activating protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling molecule 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1.

[0137] The target and / or retained antigens may be tumor antigens expressed on tumor cells. Tumor antigens are well known in the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 This includes E7, ITGA2, ITGA3, SLC39A6, MAGE, Mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-Cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, and WT1.

[0138] The target and / or retained antigens may be immune checkpoint proteins. Examples of immune checkpoint proteins include, but are not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0139] The target and / or retaining antigens may be cell surface molecules such as proteins, lipids, or polysaccharides. In some embodiments, the target and / or retaining antigens are located on tumor cells, virus-infected cells, bacterial-infected cells, damaged erythrocytes, arterial plaque cells, inflammatory tissue cells, or fibrous tissue cells. The target and / or retaining antigens may include immune response modulators. Examples of immune response modulators include, but are not limited to, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-12 (IL-12), interleukin-15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.

[0140] The target and / or retained antigens may be cytokine receptors. Examples of cytokine receptors include type I cytokine receptors, e.g., GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor, etc.; type II cytokine receptors, e.g., IFN-alpha receptors (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptors (IFNGR1, IFNGR2), type II IL receptor, etc.; and chemokine receptors, e.g., CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, XC chemokine receptor. Examples include, but are not limited to, ion receptors; tumor necrosis receptor superfamily receptors, such as TNFRSF5 / CD40, TNFRSF8 / CD30, TNFRSF7 / CD27, TNFRSF1A / TNFR1 / CD120a, TNFRSF1B / TNFR2 / CD120b, etc.; TGF-beta receptors, such as TGF-beta receptor 1, TGF-beta receptor 2, etc.; and Ig superfamily receptors, such as IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR, etc.

[0141] Linker As described above, the pharmaceutical composition comprises one or more linker sequences. The linker sequences help to provide flexibility between polypeptides, for example, allowing a blocking portion to inhibit the activity of a cytokine polypeptide. The linker sequences can be positioned between any or all of the cytokine polypeptide, serum half-life extender elements, and / or blocking portions. As described herein, at least one of the linkers is protease-cleavable and contains one or more cleavage sites for one or more desired proteases. Preferably, the desired proteases are enriched or selectively expressed at the desired site of cytokine activity (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the site of desired cytokine activity.

[0142] Appropriate linkers can be of varying lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 1 amino acid to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0143] The orientation of components in a pharmaceutical composition is primarily a matter of design choice, and it is recognized that multiple orientations are possible, and that all of them are intended to be encompassed by this disclosure. For example, the blocking moiety can be located at the C-terminus or N-terminus of a cytokine polypeptide.

[0144] Proteases known to be associated with affected cells or tissues include serine proteases, cysteine ​​proteases, aspartate proteases, threonine proteases, glutamate proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, cathepsins B, C, D, E, K, L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, and subtilis. Syn-like protease, actinidine, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matryptase, regmine, prasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloprotease (MMP), MMP1, MMP2 Examples of proteases capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequence provided herein may be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A disingrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. MMPs may be, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9).

[0145] Table 1 shows proteases useful in the methods disclosed herein, and Table 1a shows exemplary proteases and their cleavage sites. Table 1. Proteases related to inflammation and cancer [Table 1-1] [Table 1-2] [Table 1-3] Table 1a: Exemplary proteases and protease recognition sequences [Table 1a-1] [Table 1a-2]

[0146] This specification provides pharmaceutical compositions comprising polypeptide sequences. As with all peptides, polypeptides, and proteins (including their fragments), it is understood that additional modifications may occur in the amino acid sequence of a chimeric polypeptide (amino acid sequence variant) that do not alter the properties or function of the peptide, polypeptide, or protein. Such modifications include conservative amino acid substitutions, which are discussed in further detail below.

[0147] The compositions provided herein have desired functions. Each composition comprises at least an IL-2 polypeptide, a blocking moiety, e.g., a sterically blocking polypeptide, and an optional serum half-life extender, and an optional target-directed polypeptide, and one or more linkers connecting each polypeptide in the composition. A first polypeptide, e.g., an IL-2 mutant protein, is provided as the active substance. A blocking moiety is provided to block the activity of interleukin. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed at the intended target of the active substance. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding to the interleukin polypeptide. In some embodiments, the blocking moiety, e.g., a sterically blocking peptide, is linked to interleukin via a protease-cleavable linker and cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full activity of the cytokine at the desired site.

[0148] The protease cleavage site may be a naturally occurring protease cleavage site or an artificially manipulated protease cleavage site. An artificially manipulated protease cleavage site can be cleaved by two or more proteases specific to a desired environment in which cleavage occurs, such as a tumor. The protease cleavage site may be cleavable by at least one protease, at least two proteases, at least three proteases, or at least four proteases.

[0149] In some embodiments, the linker is glycine-glycine, a saltase recognition motif, or a saltase recognition motif and a peptide sequence (Gly4Ser) n (Sequence number 126) or (Gly3Ser) n(SEQ ID NO: 127) (where n is 1, 2, 3, 4, or 5). In one embodiment, the saltase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 125), where X is any amino acid. In one embodiment, the covalent bond is between a reactive lysine residue bound to the C-terminus of the cytokine polypeptide and a reactive aspartate bound to the N-terminus of the blockage or other portion. In one embodiment, the covalent bond is between a reactive aspartate residue bound to the N-terminus of the cytokine polypeptide and a reactive lysine residue bound to the C-terminus of the blockage or other portion.

[0150] Cutting and induction As described herein, the activity of the cytokine polypeptide is attenuated in the fusion protein, and protease cleavage at a desired site of activity, such as within the tumor microenvironment, releases a form of cytokine from the fusion protein, which is far more active as a cytokine receptor agonist than the fusion protein itself. For example, the cytokine receptor activating (agonist) activity of the fusion polypeptide may be at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times smaller than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity. The cytokine polypeptide that is part of the fusion protein exists as a separate molecular entity if that molecular entity contains substantially the same amino acids as the cytokine polypeptide, substantially no additional amino acids, and is not associated with other molecules (covalently or non-covalently). If necessary, the cytokine polypeptide as a separate molecular entity may contain several additional amino acid sequences, such as tags or short sequences, to aid in expression and / or purification.

[0151] In other examples, the cytokine receptor activating (agonist) activity of a fusion polypeptide is at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times smaller than the cytokine receptor activating activity of a polypeptide containing cytokine polypeptides produced by cleavage of protease-cleavable linkers in the fusion protein. In other words, the cytokine receptor activating (agonist) activity of a polypeptide containing cytokine polypeptides produced by cleavage of protease-cleavable linkers in the fusion protein is at least about 10 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times larger than the cytokine receptor activating activity of the fusion protein.

[0152] polypeptide substitution Polypeptides described herein may contain components (e.g., cytokines, blockade regions) having the same amino acid sequence as the corresponding naturally occurring proteins (e.g., IL-2, IL-15, HSA), or may have a different amino acid sequence from the naturally occurring proteins, as long as the desired function is maintained. It is understood that one way of defining any known modifications and derivatives or possible occurrences of the disclosed proteins and encoding nucleic acids is by defining sequence variants in terms of identity with respect to a particular known reference sequence. Specifically, polypeptides and nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity with respect to the chimeric polypeptides provided herein. For example, the present specification provides polypeptides or nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, and 99 percent identity with respect to any of the nucleic acid or polypeptide sequences described herein. Those skilled in the art will readily understand how to determine the identity of two polypeptides or two nucleic acids. For example, identity can be calculated after aligning the two sequences to their highest level of identity.

[0153] Another method for calculating identity can be performed by publicly available algorithms. Optimal alignment of sequences for comparison may be performed by the local identity algorithm of Smith and Waterman, Adv.Appl.Math.2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J.Mol.Biol.48:443 (1970), the similarity search method of Pearson and Lipman, Proc.Natl.Acad.Sci.USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by check-and-verify.

[0154] The same kind of identity can be obtained for nucleic acids by algorithms disclosed, for example, Zuker, Science 244:48-52 (1989), Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989), and Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference, at least for materials related to nucleic acid alignment. It is understood that any of these methods may typically be used, and that in some cases the results of these various methods may differ, but those skilled in the art will understand that if identity is found by at least one of these methods, the sequence is said to have descriptive identity and be disclosed herein.

[0155] Protein modifications include modifications of the amino acid sequence. Amino acid sequence modifications can occur spontaneously as allele mutations (e.g., due to genetic polymorphism), due to environmental influences (e.g., due to exposure to ultraviolet light), or through human intervention (e.g., by mutagenesis of cloned DNA sequences) such as induced point mutants, deletion mutants, insertion mutants, and substitution mutants. These modifications can result in changes to the amino acid sequence, provide silent mutations, modify restriction sites, or provide other specific mutations. Amino acid sequence modifications typically fall into one or more of three categories: substitution, insertion, or deletion. Insertions include fusions at the amino and / or carboxyl termini, as well as intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than fusion insertions at the amino or carboxyl termini, for example, insertions of about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, this involves the deletion of about 2 to 6 residues at any single site within the protein molecule. Amino acid substitutions are typically single-residue substitutions, but can occur at several different locations at once; insertions are usually on about 1 to 10 amino acid residues; and deletions range from about 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., as deletions or insertions of two residues. Substitutions, deletions, insertions, or any combination thereof may be used to arrive at the final construct. Mutations should not place the sequence outside the reading frame and preferably do not create complementary regions that can generate secondary mRNA structures. Substitutional modifications are modifications in which at least one residue is removed and a different residue is inserted in its place. Such substitutions are generally made according to Table 2 below and are called conservative substitutions. Table 2. Exemplary amino acid substitutions [Table 2-1] [Table 2-2]

[0156] Modifications, including specific amino acid substitutions, are carried out by known methods. For example, modifications are performed by site-directed mutagenesis of nucleotides in the polypeptide-encoding DNA, thereby generating DNA encoding the modification, which is then expressed in recombinant cell culture. Techniques for performing substitutional mutations at specific sites in DNA with known sequences are well known, such as M13 primer mutagenesis and PCR mutation.

[0157] Modifications can be selected to optimize binding. For example, affinity maturation techniques can be used to modify scFv binding by introducing random mutations within the complementarity-determining region (CDR). Such random mutations can be introduced using various techniques, such as radiation, chemical mutagens, and error-prone PCR. Multiple rounds of mutation and selection can be performed, for example, using phage display.

[0158] This disclosure also relates to nucleic acids encoding the chimeric polypeptides described herein, and to the use of such nucleic acids for the production of chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) encoding chimeric polypeptides, and the therapeutic use of such DNA and RNA molecules.

[0159] Exemplary composition The exemplary fusion proteins of the present invention combine the above elements in various orientations. The orientations described in this section are illustrative and should not be considered limiting.

[0160] In some embodiments, the fusion protein comprises an IL-2 polypeptide, a blocking moiety, and a half-life extender. In some embodiments, the IL-2 polypeptide is positioned between the half-life extender and the blocking moiety. In some embodiments, the IL-2 polypeptide is the N-terminus relative to the blocking moiety and the half-life extender. In some such embodiments, the IL-2 polypeptide is proximal to the blocking moiety, and in some such embodiments, the IL-2 polypeptide is proximal to the half-life extender. At least one protease-cleavable linker must be included in all embodiments so that the IL-2 polypeptide can be active upon cleavage. In some embodiments, the IL-2 polypeptide is the C-terminus relative to the blocking moiety and the half-life extender. Additional elements may be linked to each other by a cleavable linker, a non-cleavable linker, or by direct fusion. In some cases, it is beneficial to include two of the same cytokine to promote dimerization.

[0161] In some embodiments, the blocking domains used can extend the half-life, and the IL-2 polypeptide is positioned between two such blocking domains.

[0162] In some embodiments, the two cytokines are contained within the same construct, with at least one being IL-2. In some embodiments, each cytokine has two blocking domains attached (three in total within one molecule), with one blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extension domains may be included to optimize pharmacokinetic properties.

[0163] In some embodiments, the three cytokines are contained within the same construct. In some embodiments, the third cytokine may function to block the other two cytokines instead of blocking the blocking domain between the other two cytokines.

[0164] Preferred half-life-extending elements for use in fusion proteins are human serum albumin (HSA), an antibody or antibody fragment that binds to serum albumin (e.g., scFV, dAb), human or humanized IgG, or any of the aforementioned fragments. In some preferred embodiments, the blocking moiety is human serum albumin (HSA), or an antibody or antibody fragment that binds to serum albumin, an antibody that binds to a cytokine to activate or prevent activation of cytokine receptor binding, another cytokine, or any of the aforementioned fragments. In preferred embodiments including an additional target-directing domain, the target-directing domain is an antibody that binds to cell surface proteins that are abundant on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin.

[0165] Treatment method and pharmaceutical composition Furthermore, the present invention provides a method for treating subjects who have or are at risk of developing diseases or disorders such as proliferative disorders, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, or graft-versus-host diseases. The method involves administering an effective amount of the fusion protein disclosed herein, typically administered as a pharmaceutical composition, to subjects in need. In some embodiments, the method further includes selecting subjects who have or are at risk of developing such diseases or disorders. The pharmaceutical composition preferably comprises a blocked cytokine, its fragment, or a mutant protein that is activated at the site of inflammation or tumor. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment, or a mutant protein, and a serum half-life extender. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment, or a mutant protein, and a blocking moiety, for example, a sterically blocking polypeptide that can sterically block the activity of the cytokine polypeptide, its fragment, or mutant protein. In yet another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, its fragment, or a mutant protein, a blocking moiety, and a serum half-life extender.

[0166] Inflammation is part of the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, and irritants, and is a defense reaction involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, remove necrotic cells and tissue damaged by the original injury and inflammatory process, and initiate tissue repair. Inflammation can arise from infection, as a symptom, or as a disease, such as cancer, atherosclerosis, allergy, myopathy, HIV, obesity, or autoimmune disease. Autoimmune diseases are chronic conditions resulting from an abnormal immune response to autoantigens. Autoimmune diseases that may be treated with polypeptides disclosed herein include, but are not limited to, lupus, celiac disease, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0167] A pharmaceutical composition may contain one or more protease-cleavable linker sequences. The linker sequences help provide flexibility between polypeptides so that each polypeptide can inhibit the activity of the first polypeptide. The linker sequences may be positioned between any or all of the cytokine polypeptides, their fragments or mutant proteins, blockage moieties, and serum half-life extension elements. Optionally, the composition may contain two, three, four, or five linker sequences. The linker sequences, two, three, or four linker sequences may be identical or different. In one embodiment, the linker sequences include GGGGS (SEQ ID NO: 132), GSGSGS (SEQ ID NO: 133), or G(SGGG)2SGGT (SEQ ID NO: 134). In another embodiment, the linker includes a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130), and SGESPAYYTA (SEQ ID NO: 131).

[0168] In some embodiments, the linker is cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, or tumor cell surface proteases.

[0169] Appropriate linkers can be of varying lengths, such as 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 1 amino acid to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0170] Furthermore, the present invention provides a method for treating subjects who have cancer or are at risk of developing it. The method comprises administering an effective amount of a chimeric polypeptide (fusion protein) disclosed herein, typically administered as a pharmaceutical composition, to a subject in need. In some embodiments, the method further comprises selecting a subject who has cancer or is at risk of developing it. The pharmaceutical composition preferably comprises a blocked cytokine, a fragment thereof, or a mutant protein that is activated at the tumor site. Preferably, the tumor is a solid tumor. The cancer may be, but is not limited to, colon cancer, lung cancer, melanoma, sarcoma, renal cell tumor, and breast cancer.

[0171] The method further includes administering one or more additional drugs to treat cancer, such as chemotherapy drugs (e.g., Adriamycin, Seruvidine, Bleomycin, Alkeran, Verban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), oncology immunotherapy drugs (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapies (e.g., CAR-T, T-cell therapy), oncolytic viruses, etc.

[0172] This specification provides pharmaceutical formulations or compositions containing a chimeric polypeptide and a pharmacologically acceptable carrier. The compositions provided herein are suitable for in vitro or in vivo administration. A pharmacologically acceptable carrier means a material that is not biologically or otherwise undesirable; that is, such material is administered to a subject without causing undesirable biological effects or interacting in a harmful manner with other components of the pharmaceutical formulation or composition in which it is contained. The carrier is selected to minimize the degradation of the active ingredient and to minimize adverse side effects in the subject.

[0173] Appropriate carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21. stThis is described in Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to make it isotonic, but the formulation may be hypertonic or hypotonic if desired. Examples of pharmacologically acceptable carriers include, but are not limited to, sterile water, physiological saline, buffers such as Ringer's solution, and dextrose solutions. The pH of the solutions is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations such as semipermeable matrices of solid hydrophobic polymers containing immunogenic polypeptides. The matrices are in the form of molded articles such as films, liposomes, or microparticles. Certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition to be administered. The carriers are suitable for the administration of chimeric polypeptides or nucleic acid sequences encoding chimeric polypeptides to humans or other subjects.

[0174] Pharmaceutical preparations or compositions are administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. Compositions are administered via one of several routes of administration, including local, oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, spray / inhalation, or by placement via bronchoscopy. In some embodiments, compositions are administered locally (non-systemically), such as in tumors, intra-articular, or intrathecal spaces.

[0175] Parenteral preparations include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol solutions / aqueous solutions, emulsions, or suspensions, and also include physiological saline and buffer media. Parenteral vehicles include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate solution, or non-volatile oils. Intravenous vehicles include liquids and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), etc. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, and inert gases, may be optionally present.

[0176] Topical formulations include ointments, lotions, creams, gels, intravenous infusions, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous bases, powder bases, or oily bases, viscosity modifiers, etc., are optionally required or desirable.

[0177] Oral administration compositions include powders or granules, suspensions or solutions dissolved in water or a non-aqueous medium, capsules, pouches, or tables. Viscosities, flavorings, diluents, emulsifiers, dispersants, or binders are optionally and preferably included.

[0178] Optionally, a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide is administered by a vector. Several compositions and methods can be used to deliver nucleic acid molecules and / or polypeptides to cells in vitro or in vivo, for example, via an expression vector. These methods and compositions can be broadly classified into two types: viral delivery systems and non-viral delivery systems. Such methods are well known in the art and are readily adaptable for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete an encoded chimeric polypeptide, or for therapeutic delivery of nucleic acids. The components of the chimeric nucleic acids disclosed herein are typically functionally linked in frame to encode a fusion protein.

[0179] As used herein, a plasmid or viral vector is a substance containing a promoter that delivers the disclosed nucleic acid to a cell in an undegraded state and brings to the expression of the nucleic acid molecule and / or polypeptide in the target cell. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpesviruses, vaccinia viruses, polioviruses, Sindbis, and other RNA viruses, including those viruses having the HIV skeleton. Any viral family that shares the characteristics of these viruses and is suitable for use as a vector is also preferred. Retroviral vectors are outlined in Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), and vectors and methods for producing them are incorporated herein by reference. Construction of replication-deficient adenoviruses has been described (Berkner et al., J.Virol.61:1213-20 (1987), Massie et al., Mol.Cell.Biol.6:2872-83 (1986), Haj-Ahmad et al., J.Virol.57:267-74 (1986), Davidson et al., J.Virol.61:1226-39 (1987), Zhang et al., BioTechniques 15:868-72 (1993)). The advantage and use of these viruses as vectors is that while they can replicate within the initial infected cell, their inability to form new infectious viral particles limits their range of transmission to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replicating vaccinia virus vectors and host-specific non-replicating vaccinia virus vectors.

[0180] The polypeptides and / or nucleic acid molecules provided can be delivered via virus-like particles. Virus-like particles (VLPs) are composed of viral proteins (or more) derived from the structural proteins of a virus. Methods for constructing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0181] The polypeptides provided can be delivered by subviral dense bodies (DBs). DBs transport proteins to target cells by membrane fusion. Methods for constructing and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0182] The polypeptides provided can be delivered by encased aggregates. Methods for preparing and using these encased aggregates are described in International Publication No. WO2006 / 110728.

[0183] Nonviral delivery methods may include expression vectors containing nucleic acid sequences encoding nucleic acid molecules and polypeptides, where the nucleic acid is functionally linked to an expression regulatory sequence. Suitable vector backbones include, for example, plasmids, artificial chromosomes, BACs, YACs, or PACs, which are commonly used in the art. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response sequences, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to create chimeric polypeptides by expression in suitable host cells, such as CHO cells.

[0184] Preferred promoters that control transcription from a vector in mammalian host cells may be obtained from a variety of sources, for example, from the genomes of viruses such as polyomaviruses, Simian virus 40 (SV40), adenoviruses, retroviruses, hepatitis B virus, and most preferably cytomegalovirus (CMV); or from heterologous mammalian promoters, such as the β-actin promoter or the EF1α promoter; or from hybrid or chimeric promoters (e.g., a CMV promoter fused to the β-actin promoter). Of course, promoters from host cells or related species are also useful herein.

[0185] An enhancer generally refers to a DNA sequence whose distance from the functional transcription start site is not constant, and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be located within introns and even within the coding sequence itself. They are typically 10–300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response sequences that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), and typically, enhancers from eukaryotic viruses are used for general expression. Preferred examples include the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer.

[0186] Promoter and / or enhancer regions may be inducible (e.g., chemically or physically modulated). Chemically modulated promoters and / or enhancers may be modulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically modulated promoters and / or enhancers may be modulated, for example, by environmental factors such as temperature and light. Optionally, promoter and / or enhancer regions may act as constitutive promoters and / or enhancers to maximize the expression of the region of the transcription unit being transcribed. In certain vectors, promoter and / or enhancer regions may be active in a cell-type-specific manner. Optionally, in certain vectors, promoter and / or enhancer regions may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type include the CMV promoter, SV40 promoter, β-actin promoter, EF1α promoter, and retroviral long terminal repeats (LTRs).

[0187] Vectors may also include, for example, origins of replication and / or markers. Marker genes can confer selectable phenotypes to cells, such as antibiotic resistance. Marker products are used to determine whether the vector has been delivered to cells and, after delivery, whether it is being expressed. Examples of selective markers for mammalian cells include dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blastocydin. If such selective markers are successfully transferred to mammalian host cells, the transformed mammalian host cells can survive under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Furthermore, expression vectors may include tag sequences designed to facilitate the manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG® tag (Kodak; New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either a carboxyl terminus or an amino terminus.

[0188] As used herein, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used herein to mean the same thing as an amino acid sequence. It should be recognized herein that the term polypeptide is not used to suggest a particular size or number of amino acids that make up a molecule, and that the peptides of the present invention may contain at most a few or more amino acid residues. As used throughout, subjects may be vertebrates, more specifically mammals (e.g., humans, horses, cats, dogs, cattle, pigs, sheep, goats, mice, rabbits, rats, and guinea pigs), birds, reptiles, amphibians, fish, and any other animals. Such terms do not imply a particular age or sex. Therefore, it is intended that adult and neonatal subjects, regardless of sex, are included. As used herein, patient or subject may be used in the same sense and may refer to a subject having a disease or disorder (e.g., cancer). The term patient or subject includes human subjects and veterinary subjects.

[0189] Individuals at risk of developing a disease or disability may have a genetic predisposition to the disease or disability, for example, having a family history, having a gene mutation that causes the disease or disability, or showing early signs or symptoms of the disease or disability. Individuals currently having a disease or disability may have one or more symptoms of the disease or disability and may have been diagnosed with the disease or disability.

[0190] The methods and agents described herein are useful for both prophylactic and therapeutic treatment. For prophylactic use, a therapeutically effective dose of the chimeric polypeptide or chimeric nucleic acid sequence encoding the chimeric polypeptide described herein is administered to the subject before the onset of symptoms (e.g., before the obvious signs of cancer or inflammation) or during the early stages of the disease (e.g., at the time of the initial signs and symptoms of cancer or inflammation). Prophylactic administration can be carried out for several days to several years before the onset of symptoms of cancer or inflammation. Prophylactic administration can be used, for example, in the prophylactic treatment of a subject diagnosed with a genetic predisposition to cancer. In therapeutic treatment, a therapeutically effective dose of the chimeric polypeptide or chimeric nucleic acid sequence encoding the chimeric polypeptide described herein is administered to the subject after the diagnosis or onset of cancer or inflammation (e.g., autoimmune disease). Prophylactic use may also be applied when the patient is receiving treatment in which inflammation is anticipated, such as chemotherapy.

[0191] In accordance with the methods taught herein, subjects are administered an effective dose of the drug (e.g., a chimeric polypeptide). The terms effective dose and effective dosage are used interchangeably. The term effective dose is defined as any amount necessary to produce a desired physiological response. The effective dose and schedule for administering the drug may be determined empirically, and making such determinations is within the scope of the skill of those skilled in the art. The dosage range for administration is sufficiently large to produce the desired effect, in which one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. Generally, the dosage may vary depending on age, condition, sex, type of disease, severity of disease or disorder, route of administration, or whether other drugs are included in the regimen, and may be determined by those skilled in the art. The dosage may be adjusted by individual physicians if there are contraindications. The dosage may vary, and more than one dose may be administered daily, for one day, or for several days. Guidelines for appropriate dosages for a given class of pharmaceuticals can be found in the literature.

[0192] As used herein, the terms treatment, cure, or cure mean a method of reducing the effects of a disease or condition or the symptoms of that disease or condition. Therefore, in the manner of disclosure, treatment may mean a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or the symptoms of that disease or condition. For example, a method for treating a disease is considered a treatment if it results in a 10% reduction in one or more symptoms of the disease in question compared to a control. Therefore, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction between 10% and 100% compared to the natural or control level. Treatment is understood not to necessarily mean a cure or complete elimination of a disease, condition, or the symptoms of that disease or condition.

[0193] As used herein, the terms "preventing," "preventing," and "prevention" mean an action, such as administering a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, before or approximately simultaneously with the onset of one or more symptoms of the disease or disorder, that inhibits or delays the onset or exacerbation of one or more symptoms of the disease or disorder. As used herein, references to reduction, reduction, or inhibition include changes of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more compared to a control level. Such terms may, but are not necessarily, include complete elimination.

[0194] Compared to IL2Rβγ, IL-2 mutants that are selective for IL2Rαβγ have been developed (Shanafelt, AB, et al., 2000, Nat Biotechnol. 18:1197-202, Cassell, DJ, et al., 2002, Curr Pharm Des., 8:2171-83). These mutants have amino acid substitutions that reduce their affinity for IL2Rβ. Since IL-2 has an undetectable affinity for IL2Rγ, these mutants consequently have low affinity for the IL2Rβγ receptor complex and a low ability to activate IL2Rβγ-expressing cells, but they retain their ability to bind to IL2Rα and to bind to and activate the IL2Rαβγ receptor complex.

[0195] One of these variants, IL2 / N88R (Bay 50-4798), was clinically tested as a low-toxicity IL-2 immune system stimulant, based on the hypothesis that NK cells expressing IL2Rβγ are the primary cause of toxicity. Bay 50-4798 was shown to selectively stimulate the proliferation of activated T cells compared to NK cells and was evaluated in Phase I / II clinical trials in cancer patients (Margolin, K., et.al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, RT, et.al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials showed that Bay 50-4798 was considerably safer and more tolerable than aldesleukin, and also demonstrated an increase in the levels of CD4+CD25+ T cells, a cell population rich in Treg cells. Following these studies, research in this field has further established the uniqueness of Treg cells and demonstrated that Treg cells selectively express IL2Rαβγ (as outlined in Malek, TR, et al., 2010, Immunity, 33:153-65).

[0196] Furthermore, it is possible to create mutants that selectively alter the affinity for the CD25 chain compared to natural IL-2.

[0197] By manipulating IL-2, it is possible to create mutants that either bind to the IL-2R complex in general, or specifically bind to the IL-2Rα subunit with a different affinity than the corresponding wild-type IL-2 or the currently available mutant (called C125S, in which the cysteine ​​residue at position 125 is replaced with a serine residue).

[0198] Therefore, the present invention features a mutant interleukin-2 (IL-2*) polypeptide that contains an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 95%, 97%, 98%, or 99% identical) to wild-type IL-2, and that binds more highly to the IL-2 trimer receptor than to the dimeric IL-2 receptor compared to WT IL-2. Typically, the mutant protein also binds to the IL-2 receptor α subunit (IL-2Rα) with a higher affinity than that of wild-type IL-2. The amino acid sequence within the mutant IL-2 polypeptide may differ from Sequence ID No. 1 (UniProtKB accession number P60568) by containing (or only containing) one or more amino acid substitutions, which may be considered conserved or non-conserved substitutions. Amino acids that do not exist in nature may also be incorporated. Alternatively, or further, an amino acid sequence may differ from SEQ ID NO: 1 (which may be considered a “reference” sequence) by containing one or more amino acid residues, and by additions and / or deletions. More specifically, an amino acid sequence may differ from that of SEQ ID NO: 1 by a mutation at at least one of the following positions in SEQ ID NO: 1: 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 (or any combination thereof). As mentioned above, only one of these positions may be modified, and positions 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more (including all of them) may be modified. For example, the amino acid sequence may differ from sequence number 1 at positions 69 and 74, and may further differ at one or more of positions 30, 35, and 128.The amino acid sequence also has the following positional sets: (a) positions 64, 69, and 74; (b) positions 69, 74, and 101; (c) positions 69, 74, and 128; (d) positions 30, 69, 74, and 103; (e) positions 49, 69, 73, and 76; (f) positions 69, 74, 101, and 133; (g) positions 30, 69, 74, and 128; (h) 69 (i) 30th, 69th, 74th, and 128th; (j) 9th, 11th, 35th, 69th, and 74th; (k) 1st, 46th, 49th, 61st, 69th, and 79th; (1) 48th, 68th, 71st, 90th, 103rd, and 114th; (m) 4th, 10th, 11th, 69th, 74th, 88th, and 133rd; (n) 15th, 30th 31st, 35th, 48th, 69th, 74th, and 92nd; (O) 30th, 68th, 69th, 71st, 74th, 75th, 76th, and 90th; (p) 30th, 31st, 37th, 69th, 73rd, 74th, 79th, and 128th; (q) 26th, 29th, 30th, 54th, 67th, 69th, 74th, and 92nd; (r) 8th, 1 At positions 3, 26, 30, 35, 37, 69, 74, and 92; and at one set of (s) positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 89, it may differ from Sequence ID No. 2 (disclosed in US7569215, incorporated herein by reference). Except for the mutations at these positions, the amino acid sequence of the mutant IL-2 polypeptide may otherwise be identical to Sequence ID No. 1. Regarding specific substitutions, the amino acid sequences are as follows: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68 It may be different from sequence number 1 by having one or more of the following: D, V69A, N71T, N71A, N71R, A73V, Q74P, S75P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A, or T133N.The nomenclature used here is consistent with scientific literature nomenclature, consisting of a single letter of the amino acid in the wild type or reference sequence, followed by its position in the sequence, and then a single letter of the amino acid that replaces it. Thus, A1T represents a threonine substitution of the alanine residue at position 1. Other mutant polypeptides within the scope of the present invention include those containing variants of SEQ ID NO: 2 with substitutions at V69 (e.g., A) and Q74 (e.g., P). For example, the amino acid sequence may have the following mutation sets relative to SEQ ID NO: (a) K64R, V69A, and Q74P, (b) V69A, Q74P, and T101A, (c) V69A, Q74P, and I128T, (d) N30D, V69A, Q74P, and F103S, (e) K49E, V69A, A73V, and K76E, (f) V69A, Q74P, T101A, and T133N, (g) N30S, V69A, Q74P, and I128A, (h) V69A, Q74P, N88D, and S99P, (i)N30S, V69A, Q74P, and I128T, (j)K9T, Q11R, K35R, V69A, and Q74P, (k)A1T, M46L, K49R, E61D, V69A, and H79R, (l)K48E, E68D, N71T, N90H, F103S, and I114V, (m)S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A, (n)E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T, (o)N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H, (p)N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T, (q)N26D, N29S, N30S, K54 This may include (r)K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T, as well as (s)N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. Sequence ID No. 2 is disclosed in US7569215 and is incorporated herein by reference as an exemplary IL-2 polypeptide sequence usable in the present invention.

[0199] As described above, any of the mutant IL-2 polypeptides disclosed herein may include, be limited to, the described sequences, or otherwise be identical to SEQ ID NO: 1. Furthermore, any of the mutant IL-2 polypeptides described herein may optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine), and / or optionally include a deletion of the alanine residue at position 1 of SEQ ID NO: 1.

[0200] The mutant IL-2 polypeptide disclosed herein has a KRα subunit with a KRα of less than approximately 28 nM. d Binding can occur at concentrations (e.g., less than approximately 25 nM, less than approximately 5 nM, about 1 nM, less than approximately 500 pM, or less than approximately 100 pM). Specifically, mutant IL-2 polypeptides may have affinity equilibrium constants of less than 1.0 nM (e.g., about 0.8 nM, 0.6 nM, 0.4 nM, or 0.2 nM). Affinity can also be expressed as the relative dissociation rate from the IL-2Rα subunit or the IL-2 receptor complex (e.g., a complex expressed on the cell surface or, in other cases, bound to the membrane). For example, mutant IL-2 polypeptides can dissociate from, for example, IL-2Rα, etc., at a lower rate compared to wild-type polypeptides or therapeutic agents using IL-2, such as IL-2*. Alternatively, affinity can be characterized as the time, or average time, that the IL-2* polypeptide persists on the surface of, for example, IL-2R, cells expressing IL-2. For example, IL-2* polypeptides can persist on the receptor for at least approximately 2 times, 5 times, 10 times, 50 times, 100 times, or 250 times (or more).

[0201] Materials, compositions, and components that may be used in, used in combination with, used in the preparation of, or are products thereof, are disclosed in relation to the methods and compositions disclosed herein. These and other materials are disclosed herein, and where combinations, subsets, interactions, groups, etc., of these materials are disclosed, specific references to various individual and aggregate combinations and permutations of these compounds may not be explicitly disclosed, but it is understood that each is specifically contemplated and described herein. For example, where a method is disclosed and considered, and several modifications that may be made to several molecules, including that method, are considered, all combinations and permutations of that method, as well as possible modifications, are specifically contemplated unless otherwise explicitly stated. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of this disclosure, including, but not limited to, steps in methods using the disclosed compositions. Therefore, where there are various additional steps that can be implemented, it is understood that each of these additional steps can be implemented using any specific step or combination of steps of the method disclosed, and that each of such combinations or subsets of combinations should be considered to be specifically contemplated and disclosed.

[0202] The publications cited herein and the materials for which they are cited are incorporated herein by reference in their entirety. [Examples]

[0203] The following are examples of the method and composition of the present invention. It will be understood that various other embodiments may be carried out based on the overview provided herein.

[0204] Example 1: Detection of IL-2, IL-2 mutant protein, IL-2Rα, and IL-2Rγ in fusion proteins by ELISA IL-2 mutant proteins are detected using commercially available antibodies, such as anti-IL-2 monoclonal antibody (JES6-1A12) (BD Pharmingen; San Jose, Calif.). A positive control is used to indicate whether the monoclonal antibody recognizes the cytokine or mutant protein. Antibodies against IL-2Rα and IL-2Rγ chains are also used. Wells of a 96-well plate are coated with PBS containing antibody (2.5 μg / ml). The wells are blocked with PBS containing 5% nonfat milk with 0.2% Tween® 20 (PBS-M-Tw), and the fusion protein is added at 37°C for 1-2 hours. After washing, anti-IL-2 biotin-labeled antibody, such as JES5H4 (BD Pharmingen), is added, and binding is detected using Strepavidin HRP (Southern Biotechnology Associates; Birmingham, Ala.). An ELISA plate was prepared by adding 50 μl of O-phenylenediamine (OPD) (Sigma-Aldrich) dissolved in 0.1 M citrate (pH 4.5) and 0.04% H2O2. The process was stopped by adding 50 μl / well of 2N H2SO4, and the absorbance at 490 nm was read.

[0205] Example 2: Protease cleavage of fusion protein by MMP9 protease Those skilled in the art will be familiar with the method for setting up a protein cleavage assay. 100 µg of protein in 1 × PBS (pH 7.4) was cleaved with 1 µg of active MMP9 (Sigma catalog no. SAE0078-50 or Enzo catalog no. BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein was then used for functional analysis or stored at -80°C before testing. The degree of cleavage was monitored by SDS-PAGE using methods well known in the art. Complete cleavage of the fusion protein by the MMP9 protease is observed, as shown in Figure 10.

[0206] Example 3: CTLL-2 assay CTLL2 cells (ATCC) were suspended and seeded at a concentration of 500,000 cells / well in a medium with or without 40 mg / ml human serum albumin (HSA), and stimulated at 37°C and 5% CO2 for 72 hours using a dilution series of recombinant hIL2 or activatable hIL2. The activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell activity was evaluated using the luminescence-based cell viability assay CellTiter-Glo (Promega). The results are shown in Figures 7-9.

[0207] Example 4: Protease cleavage of IL-2 / IL-2Rα / IL-2Rγ chimeric polypeptide leads to increased antibody accessibility and a rise in biologically active IL-2 mutant proteins. The IL-2 mutant protein fusion protein was biochemically characterized before and after cleavage using a protease, e.g., PSA. Immunoblot analysis showed that the fusion protein could be cleaved by PSA and that after sample treatment with PSA, there was an increase in the intensity of a predicted low molecular weight cleavage product of approximately 20 kDa that was reactive with anti-IL-2 antibodies. The degree of cleavage depended on the amount of PSA and the incubation time. Interestingly, analysis of the fusion protein before and after PSA treatment by ELISA revealed an increase in the apparent amount of IL-2 after PSA cleavage. In this experiment, depending on the constituent, there was an approximately 2-fold or 4-fold increase in the apparent amount of IL-2 detected using this sandwich ELISA, suggesting that antibody binding is partially hindered by the intact fusion protein. Divided samples of the same specimen were also analyzed after PSA treatment using a CTLL-2 cell line that requires IL-2 for growth and survival, and cell viability could be confirmed using a colorimetric MTT assay. In this assay, the more the supernatant can be further diluted, the higher the amount of biologically active IL-2 present, and there is an increase in the amount of biologically active IL-2 after PSA cleavage. The increase in IL-2 mutant protein suggests that after PSA cleavage, there is an increase in the amount of low molecular weight cleavage fragments of approximately 20 kDa that are reactive with the anti-IL-2 antibody, increased antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutant protein.

[0208] Example 5. In vivo delivery of protease-activating fusion protein results in reduced tumor growth. The chimeric polypeptide will be examined to determine whether it has potential biological effects in vivo. These experiments will use a system in which tumor cells injected into the peritoneal cavity rapidly and preferentially first bind to and proliferate in mammaries, a series of organized immunoaggregates found in the omentum (Gerber et al., Am.J. Pathol. 169:1739-52 (2006)). This system provides a convenient method for investigating the effect of fusion protein therapy on tumor growth, as it allows for multiple delivery of the fusion protein into the peritoneal cavity and enables analysis of tumor growth by examining dissociated omental cells. These experiments may use the Colon38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro. Omental tissue normally expresses relatively small amounts of MMP2 and MMP9, but MMP levels are increased when Colon38 tumors are present in the omentum. This tumor model will be used to investigate the ability of IL-2 mutant protein fusion proteins to affect tumor growth. Colon38 cells were injected intraperitoneally and allowed to bind and proliferate for one day, after which they were treated daily with fusion proteins intraperitoneally. On day 7, the animals were sacrificed, and the general tumor growth was examined using flow cytometry and colony formation assays.

[0209] Example 6: Construction of an exemplary activatable IL2 protein targeting CD20 Generation of activatable IL2 domains An IL-2 polypeptide capable of binding to the CD20 polypeptide present in tumors or tumor cells is prepared as follows: (1) a nucleic acid sequence encoding the IL-2 polypeptide sequence, and (2) a nucleic acid sequence of one or more polypeptide linkers are generated. The activatable interleukin plasmid construct can have an optional Flag, His, or other affinity tag, and is electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include T cell activation assays using T cells responsive to IL-2 stimulation in the presence of proteases.

[0210] Generation of scFv CD20 binding domains CD20 is a cell surface protein found on B lymphocytes. The CD20 antigen is present in normal and malignant pre-B lymphocytes and mature B lymphocytes, including those found in over 90% of B-cell non-Hodgkin lymphomas (NHL). The antigen is not found in hematopoietic stem cells, activated B lymphocytes (plasma cells), or normal tissues. Therefore, several antibodies, primarily derived from mice, namely 1F5, 2B8 / C2B8, 2H7, and 1H4, have been described.

[0211] Therefore, a human anti-CD20 antibody or a humanized anti-CD20 antibody is used to generate an scFv sequence of the CD20-binding domain of an activatable interleukin protein. DNA sequences encoding the human or humanized VL and VH domains are obtained, and the codons of the construct are optionally optimized for expression in Homo sapiens cells. The order of appearance of the VL and VH domains in the scFv is altered (i.e., VL-VH or VH-VL orientation), and three copies (G4S) of the subunit "G4S" or "G4S" 3 link the variable domains to construct the scFv domain. The anti-CD20 scFv plasmid construct can have an optionally chosen Flag, His, or other affinity tag, and is electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.

[0212] Cloning of DNA expression constructs encoding an activatable IL2 protein An activatable interleukin protein is constructed using an activatable IL2 construct containing a protease cleavage domain, combined with an anti-CD20 scFv domain and a serum half-life extension element (e.g., HSA-binding peptide or VH domain). For the expression of the activatable interleukin protein in CHO cells, the coding sequences of all protein domains are cloned into a mammalian expression vector system. In short, the gene sequences encoding the activatable interleukin domain, the serum half-life extension element, and the CD20 binding domain are synthesized separately with peptide linkers L1 and L2 and subcloned. The resulting constructs are then linked together in the order CD20-binding domain-L1-IL2 subunit 1-L2-protease cleavage domain-L3-IL2 subunit 2-L4-anti-CD20 scFv-L5-serum half-life extension element to obtain the final construct. Each expression construct is designed to contain an N-terminal signal peptide and a C-terminal hexahistidine (6×His) tag coding sequence, respectively, to facilitate protein secretion and purification.

[0213] Expression of activatable IL2 protein in stably transfected CHO cells The CHO cell expression system (Flp-In®, Life Technologies) and CHO-K1 Chinese hamster ovary cell (ATCC, CCL-61) derived cells (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4):1275-81) are used. Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0214] To adapt the cells for growth in suspension, detach them from the tissue culture flask and place them in serum-free medium. The adapted cells are then cryopreserved in a medium containing 10% DMSO.

[0215] Recombinant CHO cell lines that stably express secreted, activatable interleukin proteins are generated by transfection of suspension-adapted cells. During selection with the antibiotic hygromycin B, viable cell density is measured twice a week, and cells are centrifuged to obtain a maximum density of 0.1 × 10⁶. 6 Resuspend the viable cells / mL in fresh selective medium. Two to three weeks after selection, collect the cell pool stably expressing activatable interleukin proteins and transfer the cells to standard medium in a shaking flask. Confirm the expression of recombinant secreted proteins by performing protein gel electrophoresis or flow cytometry. Freeze-store the stable cell pool in DMSO-containing medium.

[0216] Activatable IL2 protein is produced by secretion into the cell culture supernatant during 10-day fed-batch culture of stably transfected CHO cell lines. After 10 days, the cell culture supernatant is typically collected when the culture viability exceeds 75%. Samples are taken from the produced culture every other day, and cell density and viability are evaluated. On the collection day, the cell culture supernatant is removed by centrifugation and vacuum filtered before subsequent use.

[0217] Protein expression levels and product integrity in cell culture supernatants are analyzed using SDS-PAGE.

[0218] Purification of activatable IL2 protein The activatable IL2 protein is purified from CHO cell culture supernatant in a two-step procedure. In the first step, the construct is subjected to affinity chromatography, followed by preparative size exclusion chromatography (SEC) on a Superdex 200 in the second step. The sample is buffer-exchanged and concentrated to a typical concentration of over 1 mg / mL by ultrafiltration. The purity and homogeneity of the final sample (usually over 90%) are evaluated by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting using anti-HSA or anti-idiotype antibodies, and analytical SEC, respectively. The purified protein is stored at -80°C in equal fractions until use.

[0219] Example 7: Determination of Antigen Affinity by Flow Cytometry The activatable IL2 protein of Example 6 is tested for its binding affinity to human CD20 + cells and cynomolgus monkey CD20 + cells.

[0220] A serial dilution of 100 μL of the activatable interleukin protein of Example 6 and CD20 + cells are incubated with at least one protease. After washing three times with FACS buffer, the cells are incubated on ice for 45 minutes with 0.1 mL of a 10 μg / mL mouse monoclonal anti-idiotype antibody in the same buffer. After the second wash cycle, the cells are incubated with 0.1 mL of a 15 μg / mL FITC-conjugated goat anti-mouse IgG antibody under the same conditions as before. As a control, the cells are incubated with anti-His IgG without using the activatable IL2 protein and then incubated with the FITC-conjugated goat anti-mouse IgG antibody. Next, the cells are washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1×10 4The fluorescence of living cells is measured using a Beckman-Coulter FC500 MPL flow cytometer (Beckman-Coulter, Krefeld, Germany) with MXP software or a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany) with Incyte software. The average fluorescence intensity of the cell sample is calculated using MXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values ​​of cells stained with secondary and tertiary reagents alone, the value is then calculated using the equation for determining a single-site bond (hyperbola) in GraphPad Prism (version 6.00 for Windows®, GraphPad Software, La Jolla, California USA). D Used for calculating values.

[0221] CD20 binding and cross-reactivity in human CD20 + Evaluate using tumor cell lines. Cross-reactivity K D The ratio was determined for CHO cell lines expressing recombinant human antigen or recombinant cynomolgus monkey antigen. D Perform calculations using the values.

[0222] Example 8: Cytotoxicity assay The activatable IL2 protein of Example 6, its CD20 + We will evaluate the mediation of immune responses against target cells in vitro.

[0223] Fluorescent label CD20 + REC-1 cells (mantle cell lymphoma cell line, ATCC CRL-3004) are used in the example. 6Incubate the target cells with isolated PBMCs from a random donor or CB15 T cells (standardized T cell line) as effector cells in the presence of the activatable IL2 protein and at least one protease. After incubation at 37°C for 4 hours in a humidified incubator, measure the release of the fluorescent dye from the target cells into the supernatant using a spectrofluorometer. Example 6 Target cells incubated without the activatable IL2 protein and target cells completely lysed by saponin addition at the end of incubation are used as negative and positive controls, respectively.

[0224] Based on the measured number of remaining living target cells, the percentage of specific cell lysis is calculated according to the following formula: [1 - (living target (試料) Number of living targets (自然発生的) Number of ) × 100%. Sigmoid dose-response curves and EC are calculated by nonlinear regression / 4-parameter logistic fitting using GraphPad Software. 50 The values ​​are calculated. Using the lysis values ​​obtained for a given antibody concentration, the sigmoid dose-response curve is calculated by 4-parameter logistic fit analysis using Prism software.

[0225] Example 9: Pharmacokinetics of Activatable IL2 Protein Examples 6 The activatable IL2 protein will be evaluated in animal studies based on its half-life and elimination time.

[0226] Activatable IL2 protein is administered to cynomolgus monkeys as a 0.5 mg / kg bolus injection into the saphenous vein. Another group of cynomolgus monkeys receives an IL2 construct of comparable size but lacking the serum half-life extender. The third and fourth groups receive an IL2 construct with the serum half-life extender, and a cytokine containing CD20 and the serum half-life extender, respectively, both of comparable size to the activatable interleukin protein. Each test group consists of five monkeys. Serum samples are collected and serially diluted at the indicated time points, and the protein concentration is determined using a CD20-binding ELISA.

[0227] Pharmacokinetic analysis is performed using the plasma concentration of the test substance. The group-mean plasma data for each test substance, when plotted against time after administration, follow a multi-exponential profile. The data are fitted using a standard two-compartment model with bolus dose and first-order rate constant for the distribution and elimination phases. The general equation for best fitting intravenous administration data is c(t)=Ae -αt +Be -βt The equation is given by A=D / V(α-k21) / (α-β) and B=D / V(β-k21) / (α-β), where c(t) is the plasma concentration at time t, A and B are intercepts on the Y axis, and α and β are the apparent first-order rate constants of the distribution phase and elimination phase, respectively. The α phase is the initial phase of clearance, reflecting the distribution of protein into all extracellular fluid of the animal, and the second or β phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D / V(α-k21) / (α-β) and B=D / V(β-k21) / (α-β), where α and β (if α>β) are given by a quadratic equation r 2 The roots of +(k12+k21+k10)r+k21k10=0 are used, and the estimation parameters are V=volume of distribution, k10=elimination rate, k12=transfer rate from compartment 1 to compartment 2, k21=transfer rate from compartment 2 to compartment 1, and D=dose administered.

[0228] Data analysis: Graphs of concentration vs. time profiles are created using KaleidaGraph (KaleidaGraph (trademark) V.3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values reported as less than the limit of detection (LTR) are not included in the PK analysis and are not represented in the graphs. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin (registered trademark) Professional V.3.1 WinNonlin (trademark) Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are calculated as described in Ritschel W A and Kearns G L, 1999, IN: Handbook Of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, D.C.

[0229] Example 6 The activatable IL2 protein of

[0230] Example 10: Xenograft Tumor Model Example 6 The activatable IL2 protein of

[0231] Female immunodeficient NOD / scid mice are irradiated at a sublethal dose (2 Gy) and 6 Ramos RA1 cells of 3 are subcutaneously inoculated into the right dorsal flank. When the tumors reach 100-200 mm 7Inject activated human T cells intraperitoneally. Three days later, the animals in Group 3 were subsequently treated with a total of 9 intravenous administrations (once a day for 9 days) with 50 μg of the activatable interleukin protein of Example 6 Groups 1 and 2 were treated with only the vehicle. Measure body weight and tumor volume for 30 days.

[0232] Example 6 Animals treated with the activatable IL2 protein of Example are expected to have a statistically significant delay in tumor growth compared to their respective vehicle-treated control groups.

[0233] Preferred embodiments of the invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Here, those skilled in the art will envision numerous variations, modifications, and substitutions without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in the practice of the invention. The scope of the invention is defined by the following claims, and it is intended that the methods and structures included in these claims, and their equivalents, be thereby encompassed.

[0234] Example 11: HEK Blue assay HEK-Blue IL2 cells (InvivoGen) were seeded in suspension at a concentration of 50,000 cells / well in a medium with or without 15 - 40 mg / ml human serum albumin (HSA), and stimulated at 37 °C, 5% CO2 for 24 hours using a dilution series of recombinant hIL2 or activatable hIL2. The activities of non-cleaved and cleaved activatable hIL2 were tested. Cleaved inducible hIL2 was generated by incubation with active MMP9. IL12 activity was evaluated by quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric-based assay. The results are shown in Figure 11.

[0235] Example 12: MC38 Experiment We used the MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro. Using this tumor model, we investigated the ability of the fusion protein to influence tumor growth. Example 12a: MC38 IL-2POC Medications and treatments: [Table 4-1] [Table 4-2] Example 12b: MC38 IL-2 Medications and treatments: [Table 5-1] [Table 5-2] Example 12c: Treatment with ACP16, ACP132, and ACP21 Mice were anesthetized with isoflurane to transplant cells that reduce ulcer formation. CR female C57BL / 6 mice were transplanted into the subcutaneous tissue of their flanks in 5 × 10⁻¹⁰ 5 The cells were prepared using 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL / mouse. Mice were 8-12 weeks old at the start of treatment. The tumors had an average size of 100-150 mm. 3Pair matching was performed and treatment initiated when the target was reached. ACP16 was administered at 17ug, 55ug, 70ug, or 230ug / animal; ACP132 at 9ug, 28ug, 36ug, or 119ug / animal; and ACP21 at 13ug, 42ug, 54ug, or 177ug / animal. Body weight was measured at the start and then twice weekly until the end of the treatment. Caliper measurements were also performed twice weekly until the end of the treatment. Any adverse reactions were reported immediately. Individual animals that showed a weight loss of more than 30% once, or a weight loss of more than 25% three consecutive times, were euthanized. Any group with a mean weight loss of more than 20% or a mortality rate of more than 10% was discontinued, and the group was not euthanized but allowed to recover. Within groups with a weight loss of more than 20%, individuals that reached the individual weight loss endpoint were euthanized. If group treatment related to weight loss recovered to within 10% of the original body weight, administration was resumed with a lower dose or less frequent dosing schedule. Exceptions for % weight recovery in untreated animals were permitted on an individual basis. The endpoint was delayed tumor growth (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1500 mm². 3 The endpoint was defined as either day 45 or day 45, whichever came first. Responding individuals were then tracked. Upon reaching the endpoint, the animals were euthanized. The results are shown in Figure 17. Example 12d: MC38 Re-challenge The cured mice from Example 12b (treated with ACP16) were re-challenged with tumor transplantation to determine whether antitumor memory was established from the initial treatment. Medications and treatments: [Table 6-1] [Table 6-2] procedure: Mice were anesthetized with isoflurane to transplant cells that reduce ulcer formation. This part of the experiment began on the day of transplantation (Day 1). Group 1 received 5 × 10 cells subcutaneously in the flank. 5The study consisted of 33 CR female C57BL / 6 mice prepared with 0% Matrigel containing MC38 tumor cells. Groups 2-6 were subjected to 5 × 10⁶ injections into the left flank subcutaneous tissue. 5 The study consisted of 33 CR female C57BL / 6 mice prepared with 0% Matrigel containing MC38 tumor cells. Tumors from the previous MC38 experiment (Example 12b) were transplanted into the right flank of each animal. The cell injection volume was 0.1 mL / mouse. The control mice were 14–17 weeks old at the start of the experiment. These mice were matched in age to the mice from the previous MC38 experiment (Example 12b). No active drugs were administered during the rechallenge. Body weight was measured twice a week until the end of the experiment, and caliper measurements were also performed. Any adverse reactions or deaths were reported immediately. Individual animals that showed a weight loss of more than 30% once, or a weight loss of more than 25% three times consecutively, were euthanized. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The endpoint of the experiment was a tumor volume of 1000 mm³. 3 The endpoint was defined as either day 45 or day 15, whichever came first. Responding individuals were followed up if possible. Upon reaching the endpoint, the animals were euthanized. The results are shown in Figure 15.

[0236] Example 13. A conditionally active fusion protein containing a blocking moiety that is a serum albumin-binding domain. This embodiment describes the preparation and activity of a fusion protein, preferably a cytokine, that has inductive activity, i.e., is inactive until induced, typically by separation of the blocking portion from the active portion upon cleavage of a linker between the blocking portion and the active portion. The fusion protein contains a single antibody variable domain (dAb) that binds to serum albumin via a CDR loop and binds to an active portion (here, anti-CD3 scFV) via one or more non-CDR loops (e.g., C loops). The serum albumin-binding blocking portion is functionally linked to the active portion via a protease-cleavable linker, and the active portion is functionally linked to a target-directed domain (here, anti-epidermal growth factor receptor (EGFR) dAb or anti-prostate-specific membrane antigen (PSMA) dAb) via a protease-insensitive linker. These fusion proteins can be administered as inactive proteins that are activated upon cleavage of the protease-cleavable linker, followed by the release of the inhibitory albumin-binding domain. The anti-CD3 scFV in the fusion protein is a substitute for the cytokine desired in the fusion protein described herein. Similar fusion proteins containing a desired cytokine (e.g., IL-2, IL-12, interferon) or its functional fragment or mutant protein, a target-directing domain, and an albumin-binding dAb that also binds to and inhibits the cytokine or its functional fragment or mutant protein, can be prepared using the methods described and illustrated herein. Antiserum albumin dAbs that bind to and inhibit the activity of a desired cytokine or its functional fragment or mutant protein can provide both steric masking of the cytokine (by proximity of the cytokine to the bound serum albumin) and specific masking of the cytokine (by binding to the cytokine via a non-CDR loop (e.g., C-loop)). Antiserum albumin dAbs that bind to and inhibit the activity of a desired cytokine or its functional fragment or mutant protein can be obtained using appropriate methods, for example, by introducing amino acid sequence diversity into the non-CDR loop (e.g., C-loop) of the antiserum albumin-binding dAb and screening for binding to the desired cytokine.For selection, any suitable method can be used, such as a phage display. For example, an exemplary antiserum albumin dab that can be used has the following sequence. The amino acid sequence within the C-loop (bold underlined part) is diversified (e.g., randomized), and the resulting dAb can be screened for binding to serum albumin via CDR interaction and binding to cytokines via non-CDR loop interaction. If desired, the amino acid sequence of a known cytokine-binding peptide can be introduced into the C-loop. [Chemical formula]

[0237] A. Protease activation of ProTriTAC results in a substantial enhancement of activity in vitro Recombinant active drug fragments mimicking purified ProTriTAC (prodrug), uncleavable ProTriTAC [prodrug (uncleavable)], and protease-activated ProTriTAC (active drug) were tested for binding to recombinant human CD3 in an ELISA assay, binding to purified human primary T cells in a flow cytometry assay, and functional efficacy in a T cell-dependent cytotoxicity assay.

[0238] In ELISA, the appropriate concentration of soluble ProTriTAC protein was incubated with immobilized recombinant human CD3e (R&D Systems) in PBS supplemented with 15 mg / ml human serum albumin at room temperature for 1 hour. The plate was blocked using SuperBlock (Thermo Fisher), washed using PBS containing 0.05% Tween-20, detected using a non-competitive anti-CD3 idiotypic monoclonal antibody 11D3, and then using a peroxidase-labeled secondary antibody and TMB-ELISA substrate solution (Thermo Fisher).

[0239] In flow cytometry, soluble ProTriTAC protein at the indicated concentrations was incubated with purified human primary T cells for 1 hour at 4°C in PBS containing 2% fetal bovine serum and 15 mg / ml human serum albumin. The plates were washed with PBS containing 2% fetal bovine serum, detected using AlexaFluor 647-labeled non-competitive anti-CD3 idiotype monoclonal antibody 11D3, and the data were analyzed using FlowJo 10 (FlowJo, LLC).

[0240] For functional efficacy in T cell-dependent cell-mediated cytotoxicity assays, soluble ProTriTAC protein at the indicated concentrations was incubated with purified resting human T cells (effector cells) and HCT116 cancer cells (target cells) at 37°C for 48 hours with an effector:target cell ratio of 10:1. The HCT116 target cell line was stably transfected with the luciferase reporter gene, allowing for the measurement of specific T cell-mediated cell killing using ONE-Glo (Promega).

[0241] B.ProTriTAC exhibits potent protease-dependent antitumor activity in rodent tumor xenograft models. The in vivo antitumor activity of ProTriTAC was evaluated in subcutaneous xenograft tumors of HCT116 mixed with proliferated human T cells in immunodeficient NCG mice. Specifically, on day 0, each mouse was mixed with 5 × 10⁶ HCT116 cells and 2.5 × 10⁶ proliferated T cells. ProTriTAC administration was started the following day and administered once daily for 10 days by intraperitoneal infusion. Tumor volume measurements were obtained using caliper measurements at the indicated time points and calculated using the formula V = (length × width × width) / 2.

[0242] C. Expression, purification, and stability of exemplary ProTriTAC triplicate molecules Protein production The sequence encoding the inducible fusion protein molecule was cloned into the mammalian expression vector pcDNA 3.4 (Invitrogen) after the leader sequence and before the 6× histidine tag (SEQ ID NO: 136). Expi293F cells (Life Technologies, A14527) were maintained in suspension in Optimum Growth Flasks (Thomson) at 0.2–8×1e6 cells / ml in Expi 293 medium. Expi293 cells were transfected with purified plasmid DNA according to the Expi293 Expression System Kit (Life Technologies, A14635) protocol and maintained for 4–6 days post-transfection. Alternatively, the sequence encoding the fusion protein molecule was cloned into the mammalian expression vector pDEF38 (CMC ICOS) and transfected into CHO-DG44 dhfr- cells to generate a stable pool, which was cultured in production medium for up to 12 days before purification. The amount of exemplary fusion protein in the conditional medium was quantified using an Octet RED 96 instrument (ForteBio / Pall) with a Protein A tip, using a control fusion protein for the standard curve. Conditional medium from any of the host cells was filtered and partially purified by affinity chromatography and desalting chromatography. Subsequently, the fusion protein was polished by ion exchange and formulated in additive-containing neutral buffer simultaneously with the fraction pool. Final purity was evaluated using SDS-PAGE and analytical SEC with an Acquity BEH SEC 200 1.7u 4.6×150 mm column (Waters Corporation), separated into aqueous / organic mobile phase with additives at neutral pH on a 1290 LC System, and peaks were integrated using Chemstation CDS software (Agilent). The fusion proteins purified from CHO host cells are shown in the SDS-PAGE below.

[0243] Stability evaluation The purified fusion protein was divided into two formulations and further divided into equal volumes in sterile tubes. These were then stressed by five freeze-thaw cycles, each consisting of more than one hour at -80°C and room temperature, or by one week of incubation at 37°C. The stressed samples were evaluated for concentration and turbidity using UV spectroscopy, SDS-PAGE, and analytical SEC on a UV-transmitted 96-well plate (Corning 3635) with SpectraMax M2 and SoftMaxPro software (Molecular Devices), and compared to the same analyses of unstressed control samples. Overlays of analytical SEC chromatograms of control and stressed samples for a single exemplary ProTriTAC molecule purified from 293 host cells are shown below.

[0244] The results indicate that ProTriTAC was produced in a yield comparable to that of regular TriTAC from a CHO stable pool, and that the protein remained stable after repeated freeze-thaw cycles and after one week at 37°C.

[0245] D. Demonstration of functional masking and stability of ProTriTAC in vivo in a 3-week pharmacokinetic study in cynomolgus monkeys. A single dose of PSMA-targeted ProTriTAC (SEQ ID NO: 119), non-cleavable ProTriTAC (SEQ ID NO: 120), unmasked / non-cleavable TriTAC (SEQ ID NO: 123), and protease-activated ProTriTAC mimicking the active drug (SEQ ID NO: 121) was administered intravenously to cynomolgus monkeys at a dose of 0.1 mg / kg. Plasma samples were collected at the indicated time points. ProTriTAC concentrations were determined using a ligand-binding assay with biotinylated recombinant human PSMA (R&D systems) and an MSD assay (Meso Scale Diagnostic, LLC) using sulfo-tagged anti-CD3 idiotype antibody cloning 11D3. Pharmacokinetic parameters were estimated using Phoenix WinNonlin pharmacokinetic software with a non-compartmental method consistent with the intravenous bolus administration route.

[0246] To calculate the in vivo prodrug conversion rate, the circulating blood concentration of the active drug is expressed in the following system of differential equations [where P is the concentration of the prodrug, A is the concentration of the active drug, and k]. a k is the rate of prodrug activation in circulating blood, c,P This is the clearance speed of the prodrag, and k c,A This was estimated by solving the equation [where is the clearance rate of the active drug].

number

number

[0247] Prodrugs, active drugs, and incleavable prodrug controls (k c,NCLV The clearance rate of the prodrug was empirically determined in cynomolgus monkeys. To estimate the rate of prodrug activation in the circulating blood, it was assumed that the difference in clearance rates between cleavable and incleavable prodrugs arises solely from nonspecific activation in the circulating blood. Therefore, the rate at which prodrugs are converted into active drugs in the circulating blood was estimated by subtracting the clearance rate of cleavable prodrugs from the clearance rate of incleavable prodrugs.

number

[0248] The initial concentration of the prodrug in circulating blood was determined empirically, and the initial concentration of the active drug was assumed to be zero.

[0249] Results and Discussion The results of Example 13 demonstrate that fusion proteins containing cytokines or their functional fragments or mutant proteins or polypeptides with desired therapeutic activity, such as anti-CD3 scFV, can be prepared such that the therapeutic activity is masked by a masking domain that binds to both serum albumin and the active polypeptide. The masking domain is functionally linked to the active domain via a protease-cleavable linker. The results indicate that this type of fusion protein can be administered as an inactive protein that is activated upon protease cleavage at a desired site of therapeutic activity, such as in a tumor.

[0250] The amino acid sequences of the fusion protein used in Example 13 are shown as Sequence IDs 116-123.

[0251] Table 3 shows an example of a fusion protein construct in detail. In Table 3, "L" is an abbreviation for "linker," and "cleav.link." is an abbreviation for "cleavable linker." Other abbreviations include "mIFNg" for mouse interferon-gamma (IFNg), "hAlbumin" for human serum albumin (HSA), and "mAlbumin" for mouse serum albumin. Table 3: Construct permutation table [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] Sequence List [Table 7-1] [Table 7-2] Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15 Table 7-16 Table 7-17 Table 7-18 Table 7-19 Built-in by reference

[0252] All patent and non-patent publication disclosures cited herein are incorporated in their entirety by reference for any purpose.

[0253] Other Embodiments The above disclosure may encompass multiple distinct inventions having independent utility. While each of these inventions has been disclosed in its preferred form(s), the specific embodiments disclosed and illustrated herein should not be considered in a restrictive sense, as numerous variations are possible. The subject matter of this disclosure includes all novel and non-obvious combinations and combination components of the various elements, features, functions, and / or properties disclosed herein. The following claims, in particular, point to certain combinations and combination components that are considered novel and non-obvious. Inventions embodied in other combinations and combination components of features, functions, elements, and / or properties may be claimed in this application, an application claiming priority from this application, or a related application. Such claims, whether they cover different inventions or the same invention, and whether they are broader, narrower, equal to, or different in scope from the original claims, are also considered to be included in the subject matter of the inventions of this disclosure. The present invention provides, for example, the following items: (Item 1) formula [A]-[L1]-[B]-[L2]-[D] or [A]-[L1]-[D]-[L2]-[B] or [D]-[L2]-[B]-[L1]-[A] or [B]-[L2]-[D]-[L1]-[A] or [D]-[L1]-[B]-[L1]-[A] or [B]-[L1]-[D]-[L1]-[A] [In the formula, A is an interleukin-2 (IL-2) polypeptide, B is an in vivo half-life extension factor, L1 and L2 are each independently polypeptide linkers, where L1 is a protease-cleavable polypeptide linker, and L2 is an optionally protease-cleavable polypeptide linker. D is a fusion polypeptide of the IL-2 blocking moiety, The fusion polypeptide has reduced IL-2 receptor activating activity, wherein the IL-2 receptor activating activity of the fusion polypeptide is at least about 10 times lower than the IL-2 receptor activating activity of a polypeptide containing the IL-2 polypeptide produced by cleavage of the protease-cleavable linker L1. (Item 2) a) Interleukin-2 (IL-2) polypeptide [A], b) IL-2 blocking section [D], and c) A fusion polypeptide comprising at least one of each of the protease-cleavable polypeptide linkers [L], The fusion polypeptide is characterized in that the IL-2 polypeptide and the IL-2 blocking portion are functionally linked by the protease-cleavable polypeptide linker, the fusion polypeptide has reduced cytokine receptor activation activity, and the cytokine receptor activation activity of the fusion polypeptide is at least about 10 times lower than the cytokine receptor activation activity of the polypeptide containing the cytokine polypeptide produced by cleavage of the protease-cleavable linker. (Item 3) The fusion polypeptide according to item 1 or item 2, wherein the agonist activity of the IL-2 polypeptide containing the fragment of the cleaved polypeptide is increased by at least about 50 times compared to the uncleaved fusion polypeptide. (Item 4) The fusion polypeptide described in any one of items 1 to 3, wherein the agonist activity is evaluated using a CTLL-2 proliferation assay, phospho STAT ELISA, or HEK Blue reporter cell assay. (Item 5) The non-cleaved fusion polypeptide is a fusion polypeptide according to any one of items 1 to 4, which binds to the IL-2 receptor alpha (IL-2Rα) in a manner substantially similar to that of naturally occurring IL-2. (Item 6) The fusion polypeptide according to any one of items 1 to 5, wherein the blocking portion inhibits the activation of IL-2 receptor alpha / beta / gamma (IL-2Rαβγ) and IL-2 receptor beta / gamma (IL-2Rβγ) by the IL-2 polypeptide of the non-cleaved fusion polypeptide. (Item 7) Each protease-cleavable linker polypeptide independently contains a sequence that can be cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), fibroblast-activating protein (FAP), ADAM metalloproteinase, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface proteases, as described in any one of items 1 to 6. (Item 8) The fusion polypeptide according to any one of claims 1 to 7, wherein each protease-cleavable polypeptide independently contains two or more cleavage sites for the same protease, or two or more cleavage sites that are cleaved by different proteases, or at least one of the protease-cleavable polypeptides contains cleavage sites for two or more different proteases. (Item 9) The IL-2 blocking portion is a fusion polypeptide according to any one of items 1 to 8, which is non-covalently bound to the IL-2 polypeptide. (Item 10) The fusion polypeptide described in item 9, wherein the non-covalent bond is pH-dependent. (Item 11) The IL-2 blocking portion comprises a ligand-binding domain or fragment of a congener receptor for IL-2, a single-domain antibody, Fab, or scFv that binds to the IL-2 polypeptide, or an antibody or antibody fragment that binds to the IL-2 receptor (e.g., Fab, single-domain antibody, scFv), as described in any one of items 1 to 10. (Item 12) The IL-2 blocking portion is also a half-life extending element, and is a fusion polypeptide as described in any one of items 1 to 11. (Item 13) The IL-2 blocking portion is a fusion polypeptide according to any one of items 1 to 8 or 12, which sterically blocks the agonist activity of the IL-2 polypeptide. (Item 14) The IL-2 blocking portion is human serum albumin, or an antigen-binding polypeptide that binds to human serum albumin, according to item 12 or 13, which is the fusion polypeptide described in item 12 or 13. (Item 15) The fusion polypeptide according to any one of items 1 to 14, wherein the IL-2 is freely dissociated from the IL-2 blocking portion after the protease-cleavable polypeptide linker is cleaved by the protease. (Item 16) The fusion polypeptide is a fusion polypeptide according to any one of items 1 to 15 that binds to IL-2Rα. (Item 17) A fusion polypeptide according to any one of items 1 to 16, further comprising at least one half-life extending element. (Item 18) The fusion polypeptide described in item 17, wherein the half-life-extending element is human serum albumin, or an antigen-binding polypeptide that binds to human serum albumin. (Item 19) The aforementioned half-life-extending element is immunoglobulin Fc, as described in item 17, for the fusion polypeptide. (Item 20) The IL-2 blocking portion is human serum albumin, human IgG, humanized IgG, sdAb, Fab, and scFv or fragments thereof, as described in item 13 of the fusion polypeptide. (Item 21) The IL-2 receptor activation is determined using a standard in vitro receptor activation assay, as well as a fusion polypeptide according to any one of items 1 to 20, using molar-based equivalent amounts of the IL-2 polypeptide and the fusion polypeptide. (Item 22) A fusion polypeptide according to any one of items 1 to 21, wherein IL-2 freely dissociates from the IL-2 blocking moiety and / or half-life extension element after the protease-cleavable sequence is cleaved by the protease. (Item 23) The fusion polypeptide according to any one of items 17 to 19, wherein the at least one half-life extension element is one half-life extension element or two half-life extension elements. (Item 24) L2 is a protease-cleavable polypeptide linker, a fusion polypeptide as described in any one of items 1 to 23. (Item 25) A fusion polypeptide as described in any one of items 1 to 24, wherein L1 is a substrate for the first protease and L2 is a substrate for the second protease. (Item 26) A fusion polypeptide as described in any one of items 2 to 25, further comprising a tumor-specific antigen-binding peptide. (Item 27) The fusion polypeptide according to item 26, wherein the tumor-specific antigen-binding peptide is linked to the IL-2 polypeptide by an inclementable linker. (Item 28) The fusion polypeptide according to item 26, wherein the tumor-specific antigen-binding peptide is linked to the IL-2 polypeptide, the half-life extension element, or the IL-2 blockade portion by a cleavable linker. (Item 29) The fusion polypeptide according to any one of items 1 to 28, wherein the serum half-life of the IL-2 polypeptide produced by cleavage of the protease-cleavable linker is equivalent to that of naturally occurring IL-2. (Item 30) The IL-2 polypeptide is a fusion polypeptide according to any one of items 1 to 29, comprising a deletion or substitution of a cystine residue corresponding to Cys125 of SEQ ID NO. (Item 31) A nucleic acid that encodes a polypeptide listed in any of items 1-30. (Item 32) A vector containing the nucleic acid described in item 31. (Item 33) Host cells containing the vector described in item 32. (Item 34) A method for producing a pharmaceutical composition, comprising culturing host cells described in item 33 under conditions suitable for the expression and harvesting of a desired polypeptide. (Item 35) i) an effective amount of a fusion polypeptide described in any one of items 1 to 30, and ii) a pharmacokinetically acceptable additive. (Item 36) A method for treating a tumor, comprising administering an effective amount of a fusion polypeptide described in any one of items 1 to 30 to a subject in need thereof. (Item 37) A fusion polypeptide as described in any one of items 1 to 30, for use as a pharmaceutical product. (Item 38) A fusion polypeptide as described in any one of items 1 to 30, for use in subjects requiring it in the treatment of tumors. (Item 39) A pharmaceutical composition for treating a tumor in a subject requiring such treatment, comprising, as an active ingredient, a fusion polypeptide described in any one of items 1 to 30.

Claims

1. a) Human interleukin-2 (IL-2) polypeptide [A]; b) Half-life extending element [B], wherein the half-life extending element is human serum albumin, an antigen-binding polypeptide that binds to human serum albumin, or immunoglobulin Fc; c) IL-2 blocking moiety [D], wherein the IL-2 blocking moiety [D] comprises a ligand-binding domain or fragment of a homologous receptor for the IL-2 polypeptide, an antibody that binds to the IL-2 polypeptide, or an antigen-binding fragment of an antibody; and d) Protease-cleavable polypeptide linker [L] A nucleic acid composition comprising one or more nucleic acid sequences encoding conditionally active IL-2, each comprising at least one of the following: The IL-2 polypeptide and the IL-2 blocking portion are functionally linked by the protease-cleavable polypeptide linker, the IL-2 polypeptide has at least 10 times lower IL-2 receptor activating activity than the polypeptide containing the IL-2 polypeptide produced by cleavage of the protease-cleavable polypeptide linker, and the nucleic acid composition is evaluated using a CTLL-2 proliferation assay, phosph STAT ELISA, or HEK Blue reporter cell assay, and using molar equivalents of the IL-2 polypeptide and the nucleic acid composition.

2. The nucleic acid composition according to claim 1, wherein the serum half-life of the IL-2 polypeptide produced by cleavage of the protease-cleavable polypeptide linker is equivalent to the half-life of naturally occurring IL-2.

3. The nucleic acid composition according to claim 1 or 2, wherein the IL-2 polypeptide comprises an IL-2 mutant or a mutant protein.

4. The nucleic acid composition according to any one of claims 1 to 3, further comprising a tumor-specific antigen-binding peptide, wherein the tumor-specific antigen-binding peptide is linked to one of [A], [B], or [D] by a cleavable linker.

5. The nucleic acid composition according to any one of claims 1 to 3, further comprising a tumor-specific antigen-binding peptide, wherein the tumor-specific antigen-binding peptide is linked to the IL-2 polypeptide via a non-cleavable linker, and the IL-2 polypeptide is linked to the half-life extension element or the IL-2 blocking portion via a cleavable linker.

6. The nucleic acid composition according to any one of claims 1 to 5, wherein the protease-cleavable polypeptide linker comprises at least one sequence that can be cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), fibroblast-activating protein (FAP), ADAM metalloproteinase, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease.

7. The nucleic acid composition according to any one of claims 1 to 6, wherein the IL-2 blocking portion inhibits the activation of the IL-2 receptor by the human IL-2 polypeptide.

8. The nucleic acid composition according to any one of claims 1 to 7, wherein the antibody fragment that binds to the IL-2 polypeptide is a single-domain antibody, Fab, or scFv.

9. The nucleic acid composition according to any one of claims 1 to 8, wherein the IL-2 polypeptide comprises a naturally occurring IL-2 polypeptide sequence or a functional fragment thereof.

10. The nucleic acid composition according to any one of claims 1 to 9, wherein the protease-cleavable polypeptide linker comprises a sequence that is cleaved by a cathepsin selected from the group consisting of cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, and cathepsin G.

11. The nucleic acid composition according to any one of claims 1 to 9, wherein the protease-cleavable polypeptide linker comprises a sequence that is cleaved by a matrix metalloproteinase (MMP) selected from the group consisting of MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, and MMP14.

12. The nucleic acid composition according to any one of claims 1 to 11, wherein the IL-2 polypeptide produced by cleavage of the protease-cleavable polypeptide linker does not contain the half-life extension element or the IL-2 blocking moiety.

13. The nucleic acid composition according to any one of claims 1 to 11, wherein the homologous receptor for IL-2 is the alpha chain of the IL-2 receptor (IL-2Rα), the beta chain of the IL-2 receptor (IL-2Rβ), or the gamma chain of the IL-2 receptor (IL-2Rγ).

14. The nucleic acid composition according to any one of claims 1 to 13, wherein the IL-2 polypeptide comprises the amino acid sequence of residues 1 to 133 of SEQ ID NO: 49, or an amino acid sequence that is at least 90% identical to amino acid residues 1 to 133 of SEQ ID NO:

49.

15. The half-life extension element is a single-domain antibody fragment (sdAb) comprising CDR1, CDR2, and CDR3; where, The CDR1 comprises the amino acid sequence of residues 174-183 of SEQ ID NO: 49; The CDR2 contains the amino acid sequence of residues 198-214 of SEQ ID NO: 49; and The CDR3 contains the amino acid sequence of residues 245-252 of SEQ ID NO:

49. The nucleic acid composition according to any one of claims 1 to 13.

16. The antibody or antigen-binding fragment comprises a heavy chain variable region; where the heavy chain variable region is CDR1 containing the amino acid sequence of residues 334-343 of SEQ ID NO: 49; CDR2 containing the amino acid sequence of residues 358-373 of SEQ ID NO: 49; and CDR3 containing the amino acid sequence of residues 404-414 of SEQ ID NO: 49 A nucleic acid composition according to any one of claims 1 to 12, comprising:

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