Activatable interleukin 12 polypeptides and methods of use thereof

Abstract

To provide activatable interleukin 12 polypeptides and methods of using the same.SOLUTION: The present disclosure features fusion proteins that are conditionally active variants of a cytokine of interest. In one aspect, the full-length polypeptides of the invention have reduced or minimal cytokine receptor activation activity, even though they contain a functional cytokine polypeptide. Upon activation, such as, for example, by cleavage of a linker that in turn links a blocking moiety, such as, for example, a steric blocking polypeptide, to the active cytokine, the cytokine can bind to its receptor and effect signal transduction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 671,225, filed May 14, 2018, U.S. Provisional Application No. 62 / 756,504, filed November 6, 2018, and U.S. Provisional Application No. 62 / 756,515, filed November 6, 2018, the entire teachings of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on May 14, 2019, is named 105365-0022_SL.txt and is 232,520 bytes in size. [Background technology]

[0003] The development of mature immunocompetent lymphoid cells from less committed precursors, their subsequent antigen-driven immune responses, and the suppression of these unwanted autoreactive responses are highly dependent on and regulated by cytokines (e.g., interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21) that utilize receptors for the common gamma chain (γc) family (Rochman et al., 2009) and family members such as IL-12, IL-18, and IL-23. IL-2 is essential for the thymic development of Treg cells and critically regulates several key aspects of mature peripheral Tregs and antigen-activated conventional T cells. IL-2 has been extensively studied due to its potent in vitro T cell growth factor activity, in part because this activity has provided a powerful tool for directly promoting immunity in patients with cancer and AIDS-HIV, or a target for antagonizing unwanted responses, such as transplant rejection and autoimmune diseases. Although in vitro studies using IL-2 provided a strong rationale for these studies, the function of IL-2 in vivo is clearly much more complex, as first demonstrated in IL-2-deficient mice, where a rapid and fatal autoimmune syndrome was observed rather than an immune deficiency (Sadlack et al., 1993, 1995). Similar observations were later made when the genes encoding IL-2Rα (Il2ra) and IL-2Rβ (Il2rb) were individually deleted (Suzuki et al., 1995; Willerford et al., 1995).

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

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

[0006] The present invention features fusion proteins that are conditionally active variants of IL-12. In one aspect, full-length polypeptides of the invention have reduced or minimal IL-12 receptor-activating activity, even though they contain functional IL-12 polypeptides. Upon activation, such as by cleavage of a linker that sequentially connects a blocking moiety, e.g., a steric-blocking polypeptide, to the active cytokine, IL-12, or a functional fragment or mutein thereof, can bind to its receptor and signal transduction. If desired, the full-length polypeptide can include a blocking polypeptide moiety that also provides additional advantageous properties. For example, the full-length polypeptide can also contain a blocking polypeptide moiety that extends serum half-life and / or directs the full-length polypeptide to a desired site of cytokine activity. Alternatively, the full-length fusion polypeptide can contain a serum half-life-extending element and / or a targeting domain that are separate from the blocking polypeptide moiety. Preferably, the fusion protein contains at least one element or domain capable of extending circulating half-life in vivo. Preferably, this element is enzymatically removed at the desired site in the body (e.g., cleaved by proteases in the tumor microenvironment), restoring pharmacokinetic properties to the payload molecule (e.g., IL2 or IFNa) substantially similar to those of the naturally occurring payload molecule. The fusion protein is then directed to the desired cell or tissue. As described herein, targeting is achieved 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 can be attached to the cytokine via a cleavable or non-cleavable linker. When attached via a non-cleavable linker, the targeting domain may further aid in retaining the cytokine within the tumor and can be considered a retention domain. The targeting domain does not necessarily need to be directly linked to the payload molecule, but may be directly linked to another element of the fusion protein. This is particularly true when the targeting domain is attached via a cleavable linker.

[0007] In one aspect, a fusion polypeptide is provided that includes an IL-12 polypeptide, or a functional fragment or mutein thereof, and a blocking moiety, such as a steric blocking domain. The blocking moiety is fused to the IL-12 polypeptide directly or via a linker and can be separated from the IL-12 polypeptide by cleavage (e.g., protease-mediated cleavage) of the fusion polypeptide at or near the fusion site or the linker or blocking moiety. For example, if the IL-12 polypeptide is fused to the blocking moiety via a linker containing a protease cleavage site, the IL-12 polypeptide is released from the blocking moiety upon protease-mediated cleavage of the linker and can bind to its receptor. The linker is designed to be cleaved at the site of desired cytokine activity, e.g., within the tumor microenvironment, to avoid off-target cytokine activity and reduce the overall toxicity of cytokine therapy.

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

[0009] In addition to the serum half-life extending element, the pharmaceutical compositions described herein preferably contain at least one or more targeting domains that bind to one or more target antigens or one or more regions on a single target antigen. It is contemplated herein that the polypeptide constructs of the present invention are cleaved at a protease cleavage site, for example, in a disease-specific microenvironment of a subject or in the blood, and that the targeting domain(s) bind to the target antigen(s) on the target cell. The 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 diseases, neoplastic diseases, inflammatory diseases, immune disorders, autoimmune diseases, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease, or host-versus-graft disease.

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

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

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

[0013] As described herein, a cytokine blocking moiety can bind to a cytokine, thereby blocking activation of the cytokine's cognate receptor.

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

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

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

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

[0018] The present disclosure also relates to the use of the conditionally active proteins, nucleic acids encoding the conditionally active proteins, vectors or host cells containing such nucleic acids for the manufacture of a medicament for treating a disease such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, graft versus host disease or host versus graft disease. [Brief explanation of the drawings]

[0019] [Figure 1-1]

[0023] Figure 1 is a schematic diagram showing a cytokine or chemokine that is activated by a protease and includes a blocking moiety. The blocking moiety may optionally function as a serum half-life extending domain. To the left of the arrow, the cytokine is connected to the blocking moiety via a protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, liberating the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 1-2]

[0023] Figure 1 is a schematic diagram showing a protease-activated cytokine or chemokine in which HSA (a blocking moiety) is directly linked to the cytokine or chemokine of interest, and a protease cleavage site is located between the HSA and the cytokine or chemokine of interest. The left side of the arrow shows that the cytokine is connected to the blocking moiety via a protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. The right side of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 1-3]

[0023] Figure 1 is a schematic diagram showing a protease-activated cytokine or chemokine with two or more HSAs (blocking moieties) directly attached to the molecule of interest. If desired, one or more of the HSAs can be attached 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 moiety via a protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. The right side of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, liberating the blocking moiety and allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties (e.g., a shorter half-life) compared to the native cytokine. [Figure 1-4]

[0023] Figure 1 is a schematic diagram showing protease-activated cytokines or chemokines, including two or more cytokines of the same or different types, each of which is linked to a binding domain via a protease-cleavable linker. To the left of the arrow, the cytokine is connected to a blocking moiety via the protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, liberating the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 2] 1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a serum half-life extending domain, connected by at least one protease-cleavable linker. To the left of the arrow, the cytokine is connected to the blocking moiety via the protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. The cytokine is also linked to a separate half-life extending element that extends its serum half-life. To the right of the arrow, in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker, thereby liberating the serum half-life extending element and the blocking moiety and allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties (e.g., a shorter half-life) compared to the native cytokine. [Figure 3]

[0023] Figure 1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, and a targeting domain, connected by at least one protease-cleavable linker. To the left of the arrow, the cytokine is connected to the blocking moiety and targeting domain via the protease-cleavable linker, thereby blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor microenvironment, a protease cleaves at the protease cleavage site in the linker, liberating the targeting domain and blocking moiety and allowing the cytokine to bind to its receptor. [Figure 4-1]

[0023] Figure 1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain, connected by at least one protease-cleavable linker, where the cytokine polypeptide and the targeting domain are connected by the protease-cleavable linker. The left side of the arrow shows that the targeting domain, blocking moiety, and half-life extending element are connected via the protease-cleavable linker(s), thereby blocking its ability to bind to its receptor. The right side of the arrow shows that in an inflammatory or tumor environment, a protease cleaves at the protease cleavage site on the linker(s), liberating the half-life extending element, targeting domain, and blocking moiety, allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties (e.g., a shorter half-life) compared to the native cytokine. [Figure 4-2]1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety, a targeting domain, and a serum half-life extending domain, all connected by at least one protease-cleavable linker. To the left of the arrow, the cytokine is connected to the targeting domain, blocking moiety, and half-life extending element via the protease-cleavable linker(s), thereby blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor environment, a protease cleaves the linker(s) at the protease cleavage site, liberating the half-life extending element and blocking moiety, allowing the cytokine to bind to its receptor. The targeting moiety remains attached, maintaining the cytokine within the tumor microenvironment. The cytokine here has similar pK properties (e.g., a shorter half-life) compared to the native cytokine. [Figure 5] 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 a V domain (CDR1, CDR2, CDR3) form a cluster at one end of a beta barrel. The CDRs are the loops connecting beta strands BC, C'-C", and FG of the immunoglobulin fold, while the lower loops connecting beta strands AB, CC', C"-D, and EF of the immunoglobulin fold, and the upper loop connecting the DE strand of the immunoglobulin fold are non-CDR loops. [Figure 6] 1 is a schematic diagram showing a protease-activated cytokine or chemokine comprising a cytokine or chemokine polypeptide, a blocking moiety that is a serum albumin-binding domain (e.g., a dAb), and a protease-cleavable linker. In the illustrated example, non-CDR loops within the serum albumin-binding domain (e.g., an sdAb) can form a binding site for the cytokine IL-12. In this example, the binding site for serum albumin can be formed by the CDRs of the serum albumin-binding domain. [Figure 7] 1 is a graph showing the results from a HEK-Blue IL-12 reporter assay performed on a human p40 / mouse p35 IL12 fusion protein before and after protease cleavage. The assay was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue® (InvivoGen). The results confirm that the IL12 protein fusion protein is active. [Figure 8-1] Figure 8 shows a series of graphs depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show greater activity in the truncated IL12 than in the intact fusion protein. The constructs tested were ACP06 (Figure 8a), ACP07 (Figure 8b), ACP08 (Figure 8c), ACP09 (Figure 8d), ACP10 (Figure 8e), and ACP11 (Figure 8f). [Figure 8-2] Figure 8 shows a series of graphs depicting the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show greater activity in the truncated IL12 than in the intact fusion protein. The constructs tested were ACP06 (Figure 8a), ACP07 (Figure 8b), ACP08 (Figure 8c), ACP09 (Figure 8d), ACP10 (Figure 8e), and ACP11 (Figure 8f). [Figure 9] The results of a protein cleavage assay are shown. The fusion protein ACP11 was run on an SDS-PAGE gel in both cleaved and uncleaved forms. As shown in the gel, cleavage was complete. [Figure 10] Schematic diagram showing a non-limiting example of an inducible cytokine protein, the construct being activated upon protease cleavage of the linker attached between the two subunits of the cytokine. [Figure 11]Figures 11a-11d are graphs showing the results from HEK-Blue assays performed on human p40 / mouse p35 IL12 fusion proteins before and after protease cleavage. The assays were performed. The results confirm that the IL12 protein fusion proteins are active. Each proliferation assay was performed with and without HSA. [Figure 12]

[0039] Figure 12 is a series of graphs showing the activity of fusion proteins in a HEKBlue IL-12 reporter assay. Figure 12a shows IL-12 / STAT4 activation in comparison with ACP11 (human p40 / mouse p35 IL-12 fusion protein) and ACP04 (negative control). Figure 12b shows a graph showing the analysis of ACP91 (chimeric IL-12 fusion protein). Circles show the activity of the control polypeptide ACP04, squares show the activity of the uncleaved ACP91 polypeptide, and triangles show the activity of the cleaved polypeptide (ACP91 + MMP9). The EC50 values ​​for each are shown in the table. Figure 12c shows a graph showing the analysis of ACP136 (chimeric IL-12 fusion protein). Circles show the activity of the control polypeptide ACP04, squares show the activity of the uncleaved ACP136 polypeptide, and triangles show the activity of the cleaved polypeptide (ACP136 + MMP9). The EC50 values ​​for each are shown in the inset table. [Figure 13-1]

[0023] Figure 1 is a series of graphs showing that truncated IL-12 polypeptides are active in the HEKBlue reporter assay. The top left panel shows that murine IL-12 is active in this assay (positive control). The bottom left and bottom right panels show the activity of the uncleaved (squares) and truncated (triangle) forms, respectively. The EC50 values ​​for each are shown in the inset, top right panel. [Figure 13-2]

[0023] Figure 1 is a series of graphs showing that truncated IL-12 polypeptides are active in the HEKBlue reporter assay. The top left panel shows that murine IL-12 is active in this assay (positive control). The bottom left and bottom right panels show the activity of the uncleaved (squares) and truncated (triangle) forms, respectively. The EC50 values ​​for each are shown in the inset, top right panel. [Figure 14]Figure 14 shows two graphs depicting the analysis of ACP31 (a murine IFNα1 fusion protein) and ACP11 (a human p40 / murine p35 IL12 fusion protein) in a tumor xenograft model. Figure 14a shows tumor volume over time in mice treated with 30 μg of ACP31 (circles), 110 μg of ACP31 (triangles), 330 μg of ACP31 (diamonds), and, as controls, 1 μg of murine wild-type IFNa1 (dashed line, squares) and 10 μg of mIFNa1 (dashed line, small circles). Vehicle alone is indicated by large open circles. The data show that tumor volume decreased over time in mice treated with ACP31 in a dose-dependent manner. Figure 14b shows tumor volume over time in mice treated with 17.5 μg of ACP11 (squares), 175 μg of ACP31 (triangles), 525 μg of ACP31 (circles), and controls of 2 μg of ACP04 (dashed line, triangles) and 10 μg of ACP04 (dashed line, diamonds). Vehicle alone is indicated by a large open circle. The data show that tumor volume decreased over time in a dose-dependent manner in both mice treated with ACP11 and ACP04 (human p40 / mouse p35 IL12 fusion protein). [Figure 15] 1 is a series of spaghetti plots showing tumor volume over time in a mouse xenograft tumor model for mice treated with vehicle alone (top left), 2 μg ACP04 (top center), 10 μg ACP04 (top right), 17.5 μg ACP11 (bottom left), 175 μg ACP11 (bottom center), and 525 μg ACP11 (bottom right). Each line represents one mouse. [Figure 16] 1 shows the properties of the TriTac polypeptide used as an example of a protease-cleavable fusion protein. [Figure 17] 1 shows the properties of the TriTac polypeptide used as an example of a protease-cleavable fusion protein. [Figure 18] 1 shows the properties of the TriTac polypeptide used as an example of a protease-cleavable fusion protein. [Figure 19] 1 shows the properties of the TriTac polypeptide used as an example of a protease-cleavable fusion protein. [Figure 20] 1 shows the properties of the TriTac polypeptide used as an example of a protease-cleavable fusion protein. [Figure 21] none. DETAILED DESCRIPTION OF THE INVENTION

[0020] Disclosed herein are methods and compositions for engineering and using constructs containing inducible IL-12. IL-12 is a potent immune agonist, leading cytokines to be considered promising therapeutic agents in oncology. However, IL-12 and other cytokines have proven to have very narrow therapeutic windows. Cytokines such as IL-12 have short serum half-lives and are considered extremely potent. As a result, therapeutic administration of cytokines results in undesirable systemic effects and toxicities. These are exacerbated by the need to administer large amounts of cytokines to achieve desired cytokine levels at the intended site of cytokine action (e.g., tumor). Unfortunately, due to cytokine biology and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in tumor treatment.

[0021] Disclosed herein are fusion proteins that overcome the problems of toxicity and short half-life that have severely limited the clinical use of IL-12 in oncology. The fusion proteins contain an IL-12 polypeptide with receptor agonist activity. However, in the context of the fusion protein, IL-12 receptor agonist activity is attenuated and circulating half-life is extended. The fusion protein contains protease cleavage sites that are cleaved by proteases associated with the desired site of IL-12 activity (e.g., tumors) and are typically enriched or preferentially 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, such as the tumor microenvironment. Cleavage by the protease at the desired site of activity, such as within the tumor microenvironment, liberates a form of IL-12 from the fusion protein that is much more active as an IL-12 receptor agonist than the fusion protein (typically at least about 100-fold more active than the fusion protein). The form of IL-12 released upon cleavage of the fusion protein typically has a short half-life, often substantially similar to that of naturally occurring IL-12, further restricting IL-12 cytokine activity to the tumor microenvironment. Although the half-life of the fusion protein is extended, the circulating fusion protein is attenuated, and toxicity is dramatically reduced or eliminated because the active cytokine is directed to the tumor microenvironment. The fusion proteins described herein, for the first time, allow for the administration of effective therapeutic amounts of cytokines to treat tumors, substantially restricting cytokine activity to the tumor microenvironment and dramatically reducing or eliminating the cytokine's undesirable systemic effects and toxicity.

[0022] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by one of ordinary skill in the art to which this invention pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ready reference, and the inclusion of such definitions herein should not be construed as necessarily representing a departure from what is generally understood in the art. The techniques and procedures described or referenced herein are generally well understood and may be readily understood by those skilled in the art using conventional methodology, e.g., Sambrook et al. These methods are commonly used using widely available molecular cloning methodologies such as those described in "Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY." Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-defined protocols and conditions unless otherwise noted.

[0023] "Cytokine" is a well-known term of art that refers to any of a group of immunoregulatory proteins (e.g., interleukins or interferons) that are secreted, inter alia, by cells of the immune system and are regulators of the immune system. Cytokine polypeptides that can be used in the fusion proteins disclosed herein include transforming growth factors such as TGF-α and TGF-β (e.g., TGF beta 1, TGF beta 2, TGF beta 3); interferons such as interferon-α, interferon-β, interferon-γ, interferon-kappa, and interferon-omega; IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, These 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 lymphotoxin; chemokines (e.g., C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), as well as fragments of such polypeptides (i.e., functional fragments of the foregoing) that activate the cytokine's cognate receptor. "Chemokine" is a term of art that refers 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 a short serum half-life, often only a few minutes. Even cytokine forms that have a modified amino acid sequence intended to extend their serum half-life but retain receptor agonist activity typically have a short serum half-life. As used herein, "short half-life cytokine" refers to a cytokine whose circulating half-life in the serum of a subject is substantially short, for example, 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 half-life than normal in the subject's body, and polypeptides that have a modified amino acid sequence intended to extend their serum half-life but retain receptor agonist activity. In this latter case, the addition of a heterologous protein domain, for example, a bona fide half-life-extending element, such as serum albumin, is not intended to include the addition of a heterologous protein domain. Typically, a short half-life cytokine polypeptide, such as an IL-12 polypeptide, has a serum half-life that is comparable to that of naturally occurring IL-12, e.g., within 5-fold, 4-fold, 3-fold or 2-fold of that of naturally occurring IL-12.

[0025] "Sortases" are transpeptidases that modify proteins by recognizing and cleaving carboxyl-terminal localization signals embedded in or attached to the terminus of target proteins or peptides. Sortase A catalyzes the cleavage of an LPXTG motif (SEQ ID NO: 80) (where X is any standard amino acid) between a Thr and a Gly residue on the target protein, resulting in transient binding of the Thr residue to an active-site Cys residue on the enzyme, forming an enzyme-thioacyl intermediate. To complete transpeptidation and create a peptide-monomer complex, a biomolecule bearing an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displacing sortase A and linking the two molecules.

[0026] As used herein, the term "steric blocker" refers to a polypeptide or polypeptide moiety that can be covalently attached directly or indirectly to a cytokine polypeptide via another moiety, such as a linker, for example, in the form of a chimeric polypeptide (fusion protein), but is not otherwise covalently attached to the cytokine polypeptide. A steric blocker can be non-covalently attached to a cytokine polypeptide, for example, via electrostatic, hydrophobic, ionic, or hydrogen bonds. A steric blocker typically inhibits or blocks the activity of the cytokine moiety due to its proximity to and comparable size to the cytokine moiety.

[0027] As used and described herein, a "half-life extending element" is a portion of a chimeric polypeptide that extends serum half-life and improves pK, for example, by modifying its size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or by modifying the parameters of absorption, biodistribution, metabolism, and elimination.

[0028] As used herein, the terms "activatable," "activate," "induce," 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 achieve activity upon cleavage of the additional element from the fusion protein.

[0029] As used herein, a "plasmid" or "viral vector" is an entity that transports a disclosed nucleic acid to a cell without degradation and contains a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the cell to which it is delivered.

[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 interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a specific size or number of amino acids comprising the molecule; the peptides of the present invention can contain up to a few or more amino acid residues.

[0031] As used throughout, a "subject" may be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects of both sexes are intended to be encompassed.

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

[0033] As used herein, the terms "treatment," "treat," or "treating" refer to a method of reducing the effects of a disease or condition or the symptoms of that disease or condition. Thus, in the disclosed methods, treatment can refer to 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 a substantially complete 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 therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or the symptoms of the disease or condition.

[0034] As used herein, the terms "prevent," "preventing," and "prevention," in reference to a disease or disorder, refer to an action, such as administering a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder, that inhibits or delays the onset or progression of one or more symptoms of the disease or disorder.

[0035] As used herein, references to "decrease," "reducing," or "inhibiting" include changes of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or more compared to appropriate control levels. Such terms can include, but do not necessarily include, complete loss of a function or property, such as, for example, agonist activity.

[0036] A "reduced cytokine receptor agonist" is a cytokine receptor agonist that has reduced receptor agonist activity compared to a naturally occurring agonist for that cytokine receptor. An attenuated cytokine agonist may have at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, at least about 1000-fold or less agonist activity compared to a naturally occurring agonist for that receptor. When a fusion protein containing a cytokine polypeptide described herein is described as being "reduced" or having "reduced activity," it is meant that the fusion protein is an attenuated cytokine receptor agonist.

[0037] An "intact fusion protein" is a fusion protein that has not had a domain removed, such as by protease cleavage. While a domain may be removable by protease cleavage or other enzymatic activity, this has not occurred when the fusion protein is "intact."

[0038] As used herein, a "moiety" refers to a portion of a molecule that has a distinct function within that molecule, such that that function can be performed by the moiety in the context of another molecule. A moiety can be a chemical substance with a specific function, or a portion of a biological molecule with a specific function. For example, a "blocking moiety" within a fusion protein is a portion of the fusion protein that can block some or all of the activity of the fusion polypeptide. It can be a protein domain such as serum albumin.

[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 against various tumor lines, but it has since been proven to have strong pro-inflammatory activity, and its overproduction can be dangerously harmful to the human body. Because systemic toxicity is a fundamental problem associated with the use of pharmacologically active amounts of cytokines in humans, novel derivatives and therapeutic strategies aimed at reducing the toxic effects of this class of biological effectors while maintaining their therapeutic efficacy are currently being evaluated.

[0040] Interleukin-12 (IL-12) is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40) that are covalently linked to generate the so-called biologically active heterodimeric (p70) molecule (Lieschke (Jana et al., 1997; Jana et al., 2014). Apart from the formation of heterodimers (IL-12 and IL-23), the p40 subunit is also secreted as a monomer (p40) and as a homodimer (p402). It is known in the art that synthesis of the heterodimer as a single chain using a linker connecting p35 to the p40 subunit preserves the full biological activity of the heterodimer. IL-12 plays an important role in the early inflammatory response to infection and in the development of Th1 cells, which favor cellular immunity. It has been shown that overproduction of IL-12 can be dangerous to the host, as it is involved in the pathogenesis of several autoimmune inflammatory diseases (e.g., MS, arthritis, type 1 diabetes).

[0041] The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of the IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T cells and natural killer cells (Trinchieri et al., 2003). IL-12Rβ1 binds to the IL-12p40 subunit, whereas IL-12p35 associated with IL-12Rβ2 confers intracellular signaling capabilities (Benson et al., 2011). Signaling through IL-12R induces the phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2), which phosphorylate and activate signal transducer and activator of transcription (STAT) 1, STAT3, STAT4, and STAT5. The specific cellular effects of IL-12 are primarily due to activation of STAT4. IL-12 induces natural killer and T cells to produce cytokines, particularly interferon (IFN)γ, which mediate many of IL-12's proinflammatory activities, such as differentiation of CD4+ T cells toward a Th1 phenotype ( Montepaone et al., 2014 ).

[0042] IL-12 is a pleiotropic cytokine whose actions form an interconnection between innate and adaptive immunity. IL-12 was first described as a factor secreted by PMA-induced EBV-transformed B cell lines. Based on its actions, IL-12 has been described as a cytotoxic lymphocyte maturation factor and natural killer cell-stimulating factor. Because of its role as a bridge between innate and adaptive immunity and its potent stimulation of IFNγ production, a cytokine that integrates natural anti-cancer defense mechanisms, IL-12 appeared to be an ideal candidate for human tumor immunotherapy. However, the severe side effects associated with systemic administration of IL-12 in clinical studies and the cytokine's very narrow therapeutic index have significantly dampened enthusiasm for its use in cancer patients (Lasek et al., 2014). Attempts to target IL-12 delivery to tumors may alleviate some of the previous problems associated with IL-12 therapy and are currently undergoing clinical trials for cancer.

[0043] The present invention is designed to address the shortcomings of direct IL-12 therapy and therapies that use other cytokines, for example, by using cytokine-blocking moieties, e.g., steric-blocking polypeptides, serum half-life extending polypeptides, targeting polypeptides, linking polypeptides such as protease-cleavable linkers, and combinations thereof. Cytokines are highly potent when administered to patients, including 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, lymphotoxin), transforming growth factors (e.g., TGF-beta1, TGF-beta2, TGF-beta3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS). As used herein, "chemokines" refer to a family of small cytokines capable of inducing chemotaxis directed toward the vicinity of responsive cells. While cytokines can provide potent therapeutics, they are associated with undesirable effects that are difficult to manage clinically, limiting their clinical use. The present disclosure relates to novel forms of cytokines that can be used in patients with reduced or eliminated undesirable effects. In particular, the present disclosure relates to pharmaceutical compositions including chimeric polypeptides (fusion proteins), nucleic acids encoding the fusion proteins, and pharmaceutical formulations of the foregoing containing cytokines or active fragments or muteins of cytokines that have reduced cytokine receptor activation activity compared to the corresponding cytokines. However, under selected conditions or in a selected biological environment, the chimeric polypeptides activate their cognate receptors, often with the same or greater potency as the corresponding naturally occurring cytokines. As described herein, this is typically achieved using cytokine-blocking moieties that block or inhibit the receptor activation function of a cytokine, its active fragment, or mutein under prevailing conditions, rather than selected conditions, such as those present at the desired site of cytokine activity (e.g., a site of inflammation or a tumor).

[0044] Chimeric polypeptides and nucleic acids encoding them can be produced using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be produced using recombinant DNA technology, synthetic chemistry, or a combination of these techniques, and expressed in a suitable expression system, such as CHO cells. Chimeric polypeptides can also be produced by expression of suitable nucleic acids, such as using synthetic or semi-synthetic chemical techniques. In some embodiments, a blocking moiety can be attached to a cytokine polypeptide via sortase-mediated conjugation. A "sortase" is a transpeptidase that modifies proteins by recognizing and cleaving carboxyl-terminal localization signals embedded in or attached to the terminus of a target protein or peptide. Sortase A catalyzes the cleavage of an LPXTG motif (SEQ ID NO: 80) (where X is any standard amino acid) between a Thr and a Gly residue on a target protein, where the Thr residue transiently binds to a Cys residue in the active site of the enzyme, forming an enzyme-thioacyl intermediate. To complete the transpeptidation and create the peptide-monomer complex, a biomolecule bearing an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displacing sortase A and linking the two molecules.

[0045] To form a cytokine blocking moiety fusion protein, a cytokine polypeptide is first tagged at its N-terminus with a polyglycine sequence or, alternatively, at its C-terminus with an LPXTG motif (SEQ ID NO: 80). The blocking moiety or other element each has a peptide attached to it, which serves as an acceptor site for the tagged polypeptide. For binding to a domain bearing an LPXTG (SEQ ID NO: 80) acceptor peptide attached via its N-terminus, the polypeptide is tagged with a polyglycine stretch at its N-terminus. For binding to a domain bearing a polyglycine peptide attached via its C-terminus, the polypeptide is tagged with an LPXTG (SEQ ID NO: 80) sortase recognition sequence at its C-terminus. Upon recognizing the polyglycine and LPXTG (SEQ ID NO: 80) sequences, the sortase forms a peptide bond between the polymeric peptide and the tagged polypeptide. The sortase reaction cleaves off a glycine residue as an intermediate and occurs at room temperature.

[0046] Various mechanisms can be utilized to eliminate or reduce the inhibition caused by the blocking moiety. For example, a pharmaceutical composition can include an IL-12 polypeptide and a blocking moiety, such as a steric blocking moiety, along with a protease-cleavable linker containing a protease cleavage site located between the IL-12 polypeptide and the cytokine blocking moiety or within the cytokine blocking moiety. Upon cleavage of the protease cleavage site, the blocking moiety can dissociate from the cytokine, which can then activate the cytokine receptor.

[0047] Any suitable linker can be used, for example, the linker can be glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 81) or (Gly3Ser) n(SEQ ID NO: 82) (where n is 1, 2, 3, 4, or 5). Typically, the sortase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 80), where X is any amino acid. In some embodiments, the covalent bond is between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of the blocker or other domain. In other embodiments, the covalent bond is between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of said blocker or other domain.

[0048] Thus, as detailed herein, the cytokine-blocking moiety (IL-12-blocking moiety) used may be a steric blocking agent. As used herein, a "steric blocking agent" refers to a polypeptide or polypeptide moiety that can be covalently bound to a cytokine polypeptide, directly or indirectly, via another moiety, such as a linker, for example, in the form of a chimeric polypeptide (fusion protein), but that is not otherwise covalently bound to the cytokine polypeptide. A steric blocking agent can be noncovalently bound to a cytokine polypeptide, for example, via electrostatic, hydrophobic, ionic, or hydrogen bonds. A steric blocking agent typically inhibits or blocks the activity of the cytokine moiety by virtue of its proximity to and comparable size to the cytokine moiety. Steric blocking of the cytokine moiety can be removed by spatially separating the cytokine moiety from the steric blocking agent, for example, by enzymatic cleavage of a fusion protein containing the steric blocking agent and cytokine polypeptide at a site between the steric blocking agent and the cytokine polypeptide.

[0049] As further detailed herein, the blocking function may be in combination with or due to the presence of additional functional components in the pharmaceutical composition, such as a targeting domain, a serum half-life extending element, and a protease-cleavable linking polypeptide, etc. For example, a serum half-life extending polypeptide may also be a steric blocker.

[0050] Various factors ensure preferential delivery and activity of IL-12 at the desired site of IL-12 activity, severely limiting systemic exposure to the interleukin through a blocking and / or targeting strategy preferentially coupled with a serum half-life extension strategy in which a blocked form of the interleukin circulates for an extended period (preferably 1-2 weeks or longer) while the activated form has a typical serum half-life of an interleukin.

[0051] In some embodiments of the invention, the half-life extending element is linked to the interleukin via a linker that is cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to liberate the full activity of the interleukin at the desired site and separate it from the non-cleavable half-life extension. In such embodiments, the fully active free interleukin has very different pharmacokinetic (pK) properties, with a half-life of hours rather than weeks. Furthermore, exposure to the active cytokine is limited to the site of desired cytokine activity (e.g., the site of inflammation or tumor), reducing systemic exposure to the active cytokine and associated toxicity and side effects.

[0052] Blocking moieties, as detailed below, can also be used to promote binding to or activation of one or more receptors, and this blockage can be relieved by removal of the blocking moiety in certain circumstances, for example, by proteolytic cleavage of a linker connecting one or more blocking moieties to the cytokine.

[0053] In another embodiment, other cytokines can be applied to improve their use, particularly as immunostimulants, e.g., for cancer therapy. 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, etc., can be tailored to maximize activation of effector cells (e.g., effector T cells, NK cells) and / or cytotoxic immune response-promoting cells at the desired site of activity, e.g., within a tumor, preferably not systemically, (e.g., induce dendritic cell maturation).

[0054] Thus, provided herein are pharmaceutical compositions comprising at least one cytokine polypeptide, such as an interleukin (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), an interferon (IFN, such as IFN-alpha, IFN-beta, and IFN-gamma), a tumor necrosis factor (e.g., TNF-alpha, lymphotoxin), a transforming growth factor (e.g., TGF-beta 1, TGF-beta 2, TGF-beta 3), a chemokine (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte-macrophage colony-stimulating factor (GM-CS), or a functional fragment or mutein of any of the foregoing. The polypeptide typically also includes at least one linker amino acid sequence, which, in certain embodiments, is susceptible to cleavage by an endogenous protease. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 24), GPLGVRG (SEQ ID NO: 83), IPVSLRSG (SEQ ID NO: 84), VPLSLYSG (SEQ ID NO: 85), or SGESPAYYTA (SEQ ID NO: 86). In other embodiments, the chimeric polypeptide further contains a blocking moiety, such as a steric-blocking polypeptide moiety, capable of blocking the activity of the interleukin polypeptide. The blocking moiety can comprise, 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 steric-blocking polypeptide moiety, which blocks the activity of the cytokine polypeptide on a cytokine receptor; in certain embodiments, the blocking moiety comprises a protease-cleavable domain. In some embodiments, blocking and reduction of cytokine activity is simply achieved by attaching additional domains to the N- or C-terminus of the interleukin domain using very short linkers. In such embodiments, blocking is expected to be relieved by protease digestion of the blocking moiety, or the short linker tethering the blocking agent to the interleukin.Once the domain is truncated or released, blocking of cytokine activity cannot be achieved.

[0055] A pharmaceutical composition, e.g., a chimeric polypeptide, can contain two or more cytokines, which can be the same or different cytokine polypeptides. For example, two or more different cytokines have complementary functions. In some examples, the first cytokine is IL-12 and the second cytokine is IL-2. In some embodiments, each of the two or more different cytokine polypeptides has an activity that regulates the activity of the other cytokine polypeptide. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine polypeptide is a T cell activating polypeptide and the second cytokine polypeptide is a non-T cell activating polypeptide. In some examples of chimeric polypeptides containing two cytokine polypeptides, the first cytokine is a chemoattractant, e.g., CXCL10, and the second cytokine is an immune cell activating polypeptide.

[0056] Preferably, the IL-12 polypeptides (including functional fragments) comprised in the fusion proteins disclosed herein are not mutated or engineered to alter the properties of the naturally occurring cytokine, such as receptor binding affinity and specificity or serum half-life, although amino acid sequence changes from the naturally occurring (including wild-type) cytokine are tolerated, for example, to facilitate cloning and to achieve desired expression levels.

[0057] Blocking part A blocking moiety can be any moiety that inhibits the ability of a cytokine to bind to and / or activate its receptor. A blocking moiety can inhibit the ability of a cytokine to bind to and / or activate its receptor by sterically blocking it and / or by covalently binding to the cytokine. Examples of suitable blocking moieties include full-length or cytokine-binding fragments or muteins of the cytokine's cognate receptor. Antibodies and fragments thereof, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (such as heavy chain variable domain (VH), light chain variable domain (VL), and variable domains of camelid nanobodies (VHH)), dAbs, etc., that bind to cytokines can also be used. Other suitable antigen-binding domains that bind to cytokines can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as those of SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of cytokines to their cognate receptors. Advantageously, such moieties also function as half-life extension elements. For example, peptides modified by conjugation 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 can also be used, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, and the like, as well as fragments and muteins of such polypeptides. For example, antibodies and antigen-binding domains that bind to proteins with long serum half-lives, such as HSA, immunoglobulins, or transferrin, or that bind to receptors that recycle to the plasma membrane, such as FcRn or the transferrin receptor, can also inhibit cytokines, particularly when bound to their antigens.Examples of such antigen-binding polypeptides include single-chain variable fragments (scFv), single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL) and variable domains of camelid nanobodies (VHH)), dAbs, etc. Other suitable antigen-binding domains that bind cytokines can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as those of SpA, GroEL, fibronectin, lipocalin, and CTLA4.

[0058] In an illustrative example, when IL-12 is the cytokine in the chimeric polypeptide, the blocking moiety can be a full-length or fragment or mutein of the first molecule of IL-12 receptor (IL-12Rβ1) or beta (IL-12Rβ2), an anti-IL-2 single domain antibody (dAb) or scFv, an anti-IL-12Rβ1 antibody or fragment thereof, an anti-IL-12Rβ2 antibody or fragment thereof, an anti-HSA dAb or scFv, and the like.

[0059] Further aspects of the invention 1. A fusion protein comprising a cytokine moiety operably linked to a binding moiety comprising a non-CDR loop and a cleavable linker, said binding moiety being capable of masking binding of said cytokine to its receptor and / or activation of said receptor by said cytokine.

[0060] 2. The fusion protein of aspect 1, wherein said binding moiety is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered bulk serum protein.

[0061] 3. The fusion protein of aspect 1 or 2, wherein said engineered scaffold comprises an sdAb, scFv, Fab, VHH, fibronectin type III domain, an immunoglobulin-like scaffold, a DARPin, a cystine-knot peptide, a lipocalin, a three-helix bundle scaffold, a protein G-related albumin binding module, or a DNA or RNA aptamer scaffold.

[0062] 4. The fusion protein of any one of aspects 1-2, wherein the binding moiety is capable of binding to a bulk serum protein.

[0063] 5. The fusion protein of any one of aspects 1 to 3, wherein said non-CDR loops are derived from a variable domain, a constant domain, a C1-set domain, a C2-set domain, an I-domain, or any combination thereof.

[0064] 6. The fusion protein of any one of aspects 1 to 4, wherein the binding moiety further comprises a complementarity determining region (CDR).

[0065] 7. The fusion protein of aspect 5, wherein the binding moiety is capable of binding to the bulk serum protein.

[0066] 8. The fusion protein of aspect 6, wherein the bulk serum protein is a half-life extending protein.

[0067] 9. The fusion protein of aspect 6 or 7, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0068] 10. The fusion protein of any one of aspects 5 to 8, wherein the CDR loops provide specific binding sites for the bulk serum protein or the immunoglobulin light chain, or any combination thereof.

[0069] 11. The fusion protein of any one of aspects 1 to 9, wherein the cleavable linker comprises a cleavage site.

[0070] 12. The fusion protein of aspect 10, wherein the cleavage site is recognized by a protease.

[0071] 13. The fusion protein of aspect 11, wherein the binding moiety is conjugated to the cytokine.

[0072] 14. The fusion protein of aspect 11 or 11, wherein the binding moiety is covalently linked to the cytokine.

[0073] 15. The fusion protein of aspect 11, 11, or 14, wherein the binding moiety is capable of masking the binding of the cytokine to its target via a specific intermolecular interaction between the binding moiety and the cytokine.

[0074] 16. The fusion protein of any one of aspects 11 to 14, wherein the non-CDR loop provides a specific binding site for binding of the moiety to the cytokine.

[0075] 17. The fusion protein of any one of aspects 11-15, wherein upon cleavage of the cleavable linker, the binding moiety is separated from the cytokine and the cytokine binds to its target.

[0076] 18. The fusion protein of any one of aspects 1 to 16, wherein the cytokine binds to a cytokine receptor.

[0077] 19. The fusion protein of aspect 17, wherein the cytokine receptor comprises 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.

[0078] 20. The fusion protein of any one of aspects 1 to 18, wherein the cleavable linker comprises a cleavage site.

[0079] 21. The fusion protein of aspect 20, wherein the cleavage site is recognized by a protease.

[0080] 22. The fusion protein of aspect 21, wherein the protease cleavage site is recognized by a serine protease, a cysteine ​​protease, an aspartic acid protease, a threonine protease, a glutamic acid protease, a metalloprotease, a gelatinase, or an asparagine peptide lyase.

[0081] 23. The protease cleavage site is selected from the group consisting of 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 a Cutinidain, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, M 22. The fusion protein of embodiment 21, wherein the fusion protein is recognized by MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target of antigen (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).

[0082] 24. A conditionally active binding protein comprising a non-CDR loop, a cytokine, and a binding moiety (M) comprising a cleavable linker (L), wherein said non-CDR loop is capable of binding to said cytokine and said binding moiety is capable of inhibiting binding of said cytokine to its receptor and / or inhibiting activation of said receptor by said cytokine.

[0083] 25. The conditionally active binding protein of aspect 24, wherein the binding moiety is capable of binding to a half-life extending protein.

[0084] 26. The conditionally active binding protein of aspect 24 or 25, wherein the binding moiety is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein.

[0085] 27. The conditionally active binding protein of embodiment 26, wherein said engineered scaffold comprises an sdAb, scFv, Fab, VHH, fibronectin type III domain, an immunoglobulin-like scaffold, a DARPin, a cystine-knot peptide, a lipocalin, a three-helix bundle scaffold, a protein G-related albumin binding module, or a DNA or RNA aptamer scaffold.

[0086] 28. The conditionally active binding protein according to any one of aspects 24-27, wherein said non-CDR loops are derived from a variable domain, a constant domain, a C1-set domain, a C2-set domain, an I-domain, or any combination thereof.

[0087] 29. The conditionally active binding protein of any one of aspects 24 to 28, wherein the binding moiety further comprises a complementarity determining region (CDR).

[0088] 30. The conditionally active binding protein of any one of aspects 24-29, wherein the binding moiety comprises a binding site specific for a bulk serum protein.

[0089] 31. The conditionally active binding protein of aspect 30, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0090] 32. The conditionally active binding protein according to any one of aspects 29 to 31, wherein said CDRs provide specific binding sites for said bulk serum protein or immunoglobulin light chain, or any combination thereof.

[0091] 33. The conditionally active binding protein of any one of aspects 29-32, wherein the binding moiety is capable of masking the binding of the cytokine to its target via a specific intermolecular interaction between the binding moiety and the cytokine.

[0092] 34. The conditionally active binding protein according to any one of aspects 29 to 33, wherein the non-CDR loop provides a specific binding site for binding of the binding moiety to the cytokine.

[0093] 35. The conditionally active binding protein of any one of aspects 24-34, wherein the cytokine binds to a cytokine receptor.

[0094] 36. The conditionally active binding protein of aspect 35, wherein said cytokine receptor comprises 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] 37. The conditionally active binding protein according to any one of aspects 24 to 36, wherein the cleavable linker comprises a cleavage site.

[0096] 38. The conditionally active binding protein according to aspect 37, wherein the cleavage site is recognized by a protease.

[0097] 39. The conditionally active binding protein according to aspect 38, wherein said protease cleavage site is recognized by a serine protease, a cysteine ​​protease, an aspartic acid protease, a threonine protease, a glutamic acid protease, a metalloprotease, a gelatinase, or an asparagine peptide lyase.

[0098] 40. The protease cleavage site is selected from the group consisting of 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 actinomycin. Nidain, bromelain, calpain, caspase, caspase-3, Mir1-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target of anti-cancer agent (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase 39. The conditionally active binding protein of embodiment 38, wherein the binding protein is recognized by type II transmembrane serine protease 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] 41. The conditionally active binding protein of aspect 24, further comprising a half-life prolonging domain attached to said binding moiety, wherein said half-life prolonging domain provides a safety switch for said binding protein, and wherein upon cleavage of said linker, separation of said binding moiety and said half-life prolonging domain from said cytokine activates said binding protein, thereby separating said binding protein from said safety switch.

[0100] 42. The conditionally active binding protein according to aspect 41, wherein said cleavage of said linker is in the tumor microenvironment.

[0101] 43. A conditionally active binding protein comprising a binding moiety that binds to a cytokine via a non-CDR loop within the binding moiety, wherein the binding moiety is further linked to a half-life prolonging domain and comprises a cleavable linker, wherein the binding protein has an extended half-life prior to its activation by cleavage of the linker, and upon activation, the binding moiety and the half-life prolonging domain are separated from the cytokine, and wherein the binding protein does not have an extended half-life in its activated state.

[0102] 44. The conditionally active binding protein according to aspect 43, wherein said cleavage of said linker is in the tumor microenvironment.

[0103] In vivo half-life extending element Preferably, the chimeric polypeptide comprises an in vivo half-life extending element. Increasing the in vivo half-life of a therapeutic molecule that naturally has a short half-life allows for a more tolerable and manageable dosing regimen without sacrificing efficacy. As used herein, a "half-life extending element" refers to a portion of a chimeric polypeptide that increases its in vivo half-life and improves its pK, for example, by modifying its size (e.g., above the renal filtration cutoff), shape, hydrodynamic radius, charge, or by modifying parameters such as absorption, biodistribution, metabolism, and elimination. An exemplary method for improving the pK of a polypeptide is by expressing elements of the polypeptide chain that bind to receptors that are recycled to the plasma membrane of cells rather than being degraded in lysosomes, such as the FcRn receptor and transferrin receptor on endothelial cells. Three proteins, such as human IgG, HSA (or fragments), and transferrin, persist in human serum much longer than would be predicted by their size alone, a function of their ability to bind to receptors that are recycled rather than being degraded in lysosomes. These proteins, or their fragments that retain FcRn binding, are routinely linked to other polypeptides to extend their serum half-life. In one embodiment, the half-life extension element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 1) can also be directly linked to the pharmaceutical composition or linked via a short linker. Fragments of HSA can also be used. HSA and its fragments can function as both blocking moieties and half-life extension elements. Human IgG can also perform similar functions.

[0104] The serum half-life extending element can also be an antigen-binding polypeptide that binds to proteins with long serum half-lives, such as serum albumin, transferrin, etc. Examples of such polypeptides include antibodies and fragments thereof, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH)), dAbs, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as those of SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0105] Some preferred serum half-life extenders are polypeptides comprising complementarity-determining regions (CDRs) and, optionally, non-CDR loops. Advantageously, such serum half-life extenders can extend the serum half-life of cytokines and also function as cytokine inhibitors (e.g., via steric blocking, non-covalent interactions, or a combination thereof) and / or as targeting 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 can be of any class or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of an Ig molecule folds into a series of parallel beta strands connected by loops. In the variable region, three of the loops constitute the "complementarity-determining regions" (CDRs) and determine the antigen-binding specificity of the molecule. An IgG molecule comprises at least two heavy (H) chains and two light (L) chains, or antigen-binding fragments thereof, interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains, CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions 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, interspersed with more conserved regions, called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments of the present disclosure, at least a portion or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of the binding moieties described herein. As shown in Figure 5, the variable domain of an immunoglobulin molecule has several beta strands 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 a V domain (CDR1, CDR2, CDR3) form a cluster at one end of a beta barrel. The CDRs are the loops connecting beta strands BC, C'-C", and FG of the immunoglobulin fold, while the lower loops connecting beta strands AB, CC', C"-D and EF of the immunoglobulin fold, and the upper loop connecting the DE strand of the immunoglobulin fold, are non-CDR loops. In some embodiments of the present disclosure, at least some amino acid residues of the constant domain, CH1, CH2, or CH3, are part of the "non-CDR loops" of the binding moiety described herein. Non-CDR loops, in some embodiments, include one or more of the AB, CD, EF, and DE loops of a C1 set domain of an Ig or Ig-like molecule; the AB, CC', EF, FG, BC, and EC' loops of a C2 set domain of an Ig or Ig-like molecule; and the DE, BD, GF, A(A1A2)B, and EF loops of an I (intermediate) set domain of an Ig or Ig-like molecule.

[0106] Within the variable domain, CDRs are thought to be involved in antigen recognition and binding, and FR residues are considered to be the scaffolding for the CDRs. However, in certain cases, some FR residues play important roles in antigen recognition and binding. Residues in framework regions that affect Ag binding are divided into two categories. First, there are FR residues that contact the antigen and are therefore part of the binding site, and some of these residues are adjacent to the CDRs. Other residues are adjacent to the CDRs but are distant from them in the 3-D structure of the molecule, such as loops in the heavy chain. Serum half-life extending domains (e.g., domains containing CDRs) can 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.

[0107] In addition to or alternatively to containing CDRs, the serum half-life extending element includes a non-CDR loop. In some embodiments, the non-CDR loop is modified to generate an antigen-binding site specific for a desired target antigen, such as a bulk serum protein such as albumin, or for a cytokine moiety or other target antigen. It is contemplated that 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 foreign to the non-CDR loop amino acid sequence, amino acid substitution, etc. In some examples, an antigenic peptide is inserted into the non-CDR loop. In some examples, the non-CDR loop is replaced with an antigenic peptide. In some cases, the modification to generate the antigen-binding site is limited to only one non-CDR loop. In other cases, two or more non-CDR loops are modified. For example, the modification is in any one of the non-CDR loops shown in Figure 5, i.e., AB, CC', C"-D, EF, and DE. In some cases, the modification is in the DE loop. In other cases, the modification is in all four loops: AB, CC', C"-D, and EF.

[0108] In some examples, the serum half-life extending element has dual binding specificity and contains a CDR that specifically binds to a bulk serum protein, 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 extending element 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 a bulk serum protein, such as serum albumin. Preferably, the serum half-life extending element inhibits binding of the cytokine domain to its cognate cytokine receptor, e.g., via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0109] In some embodiments, the serum half-life extending element directly binds to the cytokine non-covalently and inhibits its activity.

[0110] In certain examples, a binding moiety binds to a cytokine through one or more of the AB, CC', C"D, and EF loops, and binds to a bulk serum protein, such as albumin, through one or more of the BC, C'C" and FG loops. In certain examples, a binding moiety binds to a bulk serum protein, such as albumin, through its AB, CC', C"D, or EF loops, and binds to a cytokine through its BC, C'C" or FG loops. In certain examples, a binding moiety binds to a bulk serum protein, such as albumin, through its AB, CC', C"D, and EF loops, and is bound to a cytokine through its BC, C'C" and FG loops. In certain examples, a binding moiety binds to a bulk serum protein, such as albumin, through one or more of the AB, CC', C"D, and EF loops, and is bound to a cytokine through one or more of the BC, C'C" and FG loops.

[0111] The binding moiety can be any type of polypeptide. For example, in some cases, the binding moiety is a natural peptide, a synthetic peptide, or a fibronectin scaffold, or an engineered bulk serum protein. Bulk serum proteins include, for example, albumin, fibrinogen, or globulin. In some embodiments, the binding moiety is an engineered scaffold. Engineered scaffolds include, for example, sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffold (as suggested by Halaby et al., 1999. Prot Eng 12(7):563-571), DARPin, cystine-knot peptide, lipocalin, three-helix bundle scaffold, protein G-related albumin binding module, or DNA or RNA aptamer scaffold.

[0112] In some cases, the serum half-life extending element is linked to the cytokine domain via its non-CDR loop, and the cytokine domain is further connected to a targeting domain described herein. In some cases, the serum half-life extending element comprises a binding site for a bulk serum protein. In some embodiments, the CDR provides the binding site for the bulk serum protein. The bulk serum protein is, in some examples, globulin, albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor XIII, fibrinogen, IgE, or pentameric IgM. In some embodiments, the CDR forms the binding site for an immunoglobulin light chain, such as an Igκ free light chain or an Igλ free light chain.

[0113] One exemplary conditionally active protein is shown in Figure 6. In the illustrated example, non-CDR loops within a serum albumin binding domain (e.g., a dAb) can form a binding site for the cytokine IL-12. In this example, the binding site for serum albumin can be formed by the CDRs of the serum albumin binding domain.

[0114] The serum half-life extending element can be any type of binding domain, including, but not limited to, domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, and humanized antibodies. In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy chain variable domain (VH), a light chain variable domain (VL), and a variable domain of a camelid-derived nanobody (VHH). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimic such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and monobodies.

[0115] In other embodiments, the serum half-life extending element may be a water-soluble polymer or a peptide conjugated to a water-soluble polymer such as PEG. As used herein, "PEG," "polyethylene glycol," and "poly(ethylene glycol)" are synonymous and include any non-peptide, water-soluble poly(ethylene oxide). The term "PEG" also refers to a polymer containing a majority, i.e., greater than 50%, of -OCH2CH2- repeating subunits. Regarding specific configurations, PEG can have any number of different molecular weights and structures or geometries, such as "branched," "linear," "forked," "multifunctional," etc., as described in more detail below. PEG is not limited to a particular structure and may be linear (e.g., end-capped, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), or dendritic (or star-shaped) structures, each with or without one or more degradable linkages. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating tripolymers, random tripolymers, and block tripolymers. PEG can be attached 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 with 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.

[0116] Targeting and Retention Domains In certain applications, it may be desirable to maximize the time that the construct is present at its desired location in the body. This can be achieved by including an additional domain in the chimeric polypeptide (fusion protein) to affect its trafficking within the body. For example, the chimeric nucleic acid can encode a domain that targets the polypeptide to a location in the body, such as a tumor cell or an inflammatory site (this domain is referred to as a "targeting domain") and / or a domain that retains the polypeptide at a location in the body, such as a tumor cell or an inflammatory site (this domain is referred to as a "retention domain"). In some embodiments, a domain can function as both a targeting domain and a retention domain. In some embodiments, the targeting domain and / or retention domain is specific for a protease-rich environment. In some embodiments, the encoded targeting domain and / or retention domain is specific for regulatory T cells (Tregs), e.g., targets the CCR4 receptor or the CD39 receptor. Other suitable targeting and / or retention domains include those having cognate ligands overexpressed in inflamed tissue, such as the IL-1 receptor or the IL-6 receptor. In other embodiments, suitable targeting and / or retention domains include those having cognate ligands overexpressed in tumor tissue, such as Epcam, CEA, or mesothelin. In some embodiments, the targeting domain is linked to the interleukin via a linker and is cleaved at the site of action (e.g., by an inflammation- or cancer-specific protease), liberating the full activity of the interleukin at the desired site. In some embodiments, the targeting and / or retention domain is linked to the interleukin via a linker and is not cleaved at the site of action (e.g., by an inflammation- or cancer-specific protease), allowing the cytokine to remain at the desired site.

[0117] The selected antigen is optionally expressed on the surface of diseased cells or tissues, such as tumor or cancer cells. Antigens useful for tumor targeting and retention include, but are not limited to, fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), FOLR1, fibronectin EIIIB domain, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins containing two targeting and / or retention domains that bind to two different target antigens known to be expressed on diseased cells or tissues. Exemplary pairs of antigen-binding domains include, but are not limited to, EGFR / CEA, EpCAM / CEA, and HER-2 / HER-3.

[0118] Suitable targeting and / or retention domains include antigen-binding domains, e.g., antibodies and fragments thereof, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains of camelid nanobodies (VHH)), dAbs, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and binding domains based on other engineered scaffolds, such as SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of antigen-binding polypeptides include ligands for desired receptors, ligand-binding portions of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0119] In some embodiments, the targeting domain and / or retention domain specifically bind to a cell surface molecule. In some embodiments, the targeting domain and / or retention domain specifically bind to a tumor antigen. In some embodiments, the targeting polypeptide specifically and independently binds to a tumor antigen selected from at least one of fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), FOLR1, fibronectin EIIIB domain, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the targeting polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), FOLR1, fibronectin EIIIB domain, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1.

[0120] The target and / or retention antigen may be a tumor antigen expressed in tumor cells. Tumor antigens are well known in the art and include, for example, fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), FOLR1, fibronectin EIIIB domain, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and C. EA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD3 7, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 These include E6, HPV-16 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.

[0121] The target and / or retention antigen can be an immune checkpoint protein, including, but not limited to, CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM, CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8, CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA, IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.

[0122] The target and / or retention antigen can be a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the target and / or retention antigen is located on a tumor cell, a virus-infected cell, a bacteria-infected cell, an injured red blood cell, an arterial plaque cell, an inflamed tissue cell, or a fibrous tissue cell. The target and / or retention antigen can include an immune response modulator. 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.

[0123] The target and / or retention antigen may be a cytokine receptor. Examples of cytokine receptors include type I cytokine receptors, such as GM-CSF receptor, G-CSF receptor, type I IL receptor, Epo receptor, LIF receptor, CNTF receptor, TPO receptor, etc.; type II cytokine receptors, such as IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma receptor (IFNGR1, IFNGR2), type II IL receptor, etc.; chemokine receptors, such as CC chemokine receptor, CXC chemokine receptor, CX3C chemokine receptor, XC chemokine receptor, etc. 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.; Ig superfamily receptors, such as IL-1 receptor, CSF-1R, PDGFR (PDGFRA, PDGFRB), SCFR, etc., but are not limited to these.

[0124] Linker As described above, the pharmaceutical composition includes one or more linker sequences. The linker sequence serves to provide flexibility between the polypeptides, for example, allowing a blocking moiety to inhibit the activity of the cytokine polypeptide. The linker sequence can be positioned between any or all of the cytokine polypeptide, the serum half-life extending element, and / or the blocking moiety. 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 in the desired site of cytokine activity (e.g., the tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the site of the desired cytokine activity.

[0125] It will be recognized that the orientation of the components of the pharmaceutical composition is largely a matter of design choice, and that multiple orientations are possible, all of which are intended to be encompassed by the present disclosure. For example, a blocking moiety can be located at the C-terminus or N-terminus of a cytokine polypeptide.

[0126] Proteases known to be associated with affected cells or tissues include serine proteases, cysteine ​​proteases, aspartic acid proteases, threonine proteases, glutamic acid proteases, metalloproteases, aspartic peptide lyases, serum proteases, cathepsin, cathepsin B, cathepsin C, cathepsin D, cathepsin E, cathepsin K, cathepsin L, kallikrein, hKl, hK10, hK15, plasmin, collagenase, type IV collagenase, stromelysin, factor Xa, chymotrypsin-like proteases, trypsin-like proteases, elastase-like proteases, subtilisin, Synthin-like proteases, actinidain, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin, metalloexopeptidase, metalloendopeptidase, matrix metalloproteinase (MMP), MMP1, MMP2 Proteases capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequences provided herein include, but are not limited to, MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, meprin, granzyme, and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequences provided herein can be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteinase (MMP), A distigrin and metalloproteinase (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. The MMP can be, for example, matrix metalloproteinase 2 (MMP2) or matrix metalloproteinase 9 (MMP9).

[0127] Proteases useful in the methods disclosed herein are listed in Table 1, and exemplary proteases and their cleavage sites are listed in Table 1a. Table 1. Proteases associated with 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]

[0128] Pharmaceutical compositions comprising the polypeptide sequences are provided herein. As with all peptides, polypeptides, and proteins (including fragments thereof), it is understood that additional modifications may occur in the amino acid sequences of chimeric polypeptides (amino acid sequence variants) that do not alter the properties or function of the peptide, polypeptide, or protein. Such modifications include conservative amino acid substitutions, which are discussed in more detail below.

[0129] The compositions provided herein have a desired function. The compositions comprise at least an IL-12 cytokine polypeptide, a blocking moiety, e.g., a steric-blocking polypeptide, and an optional serum half-life extending element and an optional targeting polypeptide, as well as one or more linkers connecting each polypeptide in the composition. A first polypeptide, e.g., an IL-12 polypeptide, is provided as an active agent. The blocking moiety is provided to block the activity of an interleukin. A linker polypeptide, e.g., a protease-cleavable polypeptide, is provided to be cleaved by a protease specifically expressed in the intended target of the active agent. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding to the interleukin polypeptide. In some embodiments, the blocking moiety, e.g., a steric-blocking peptide, is linked to the interleukin via a protease-cleavable linker and is cleaved at the site of action (e.g., by an inflammation-specific protease) to release full cytokine activity at the desired site.

[0130] The protease cleavage site may be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site. The artificially engineered protease cleavage site can be cleaved by two or more proteases specific to the desired environment in which cleavage occurs, such as a tumor. The protease cleavage site can be cleavable by at least one protease, at least two proteases, at least three proteases, or at least four proteases.

[0131] In some embodiments, the linker is a glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly4Ser). n (SEQ ID NO: 81) or (Gly3Ser) n(SEQ ID NO: 82) where n is 1, 2, 3, 4, or 5. In one embodiment, the sortase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 80), where X is any amino acid. In one embodiment, the covalent bond is between a reactive lysine residue attached to the C-terminus of the cytokine polypeptide and a reactive aspartic acid attached to the N-terminus of the blocking moiety or other moiety. In one embodiment, the covalent bond is between a reactive aspartic acid residue attached to the N-terminus of the cytokine polypeptide and a reactive lysine residue attached to the C-terminus of the blocking moiety or other moiety.

[0132] Cleavage and inducibility As described herein, the activity of the cytokine polypeptide is attenuated in the fusion protein, and protease cleavage at the desired site of activity, such as within the tumor microenvironment, liberates a form of cytokine from the fusion protein that is significantly more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine receptor activating (agonist) activity of the fusion polypeptide may be at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold less than the cytokine receptor activating activity of the cytokine polypeptide as a separate molecular entity. A cytokine polypeptide that is part of a fusion protein exists as a separate molecular entity if that molecular entity contains substantially identical amino acids to the cytokine polypeptide, is substantially free of additional amino acids, and is not associated (covalently or noncovalently) with other molecules. Optionally, the cytokine polypeptide as a separate molecular entity may include some additional amino acid sequence, such as a tag or short sequence to aid in expression and / or purification.

[0133] In other examples, the cytokine receptor activating (agonist) activity of the fusion polypeptide is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold less than the cytokine receptor activating activity of a polypeptide containing a cytokine polypeptide produced by cleavage of a protease-cleavable linker in the fusion protein. In other words, the cytokine receptor activating (agonist) activity of a polypeptide containing a cytokine polypeptide produced by cleavage of a protease-cleavable linker in the fusion protein is at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold greater than the cytokine receptor activating activity of the fusion protein.

[0134] Polypeptide Substitution The polypeptides described herein can include components (e.g., cytokines, blocking moieties) that have the same amino acid sequence as the corresponding naturally occurring protein (e.g., IL-2, IL-15, HSA), or can have an amino acid sequence that differs from the naturally occurring protein, so long as the desired function is maintained. It is understood that one way to define any known modifications and derivatives or possible occurrences of the disclosed proteins and nucleic acids encoding them is by defining sequence variants in terms of identity to a particular known reference sequence. Specifically, disclosed are polypeptides and nucleic acids that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent identity to the chimeric polypeptides provided herein. For example, polypeptides or nucleic acids are provided that have at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identity with any of the nucleic acid or polypeptide sequences described herein.Those skilled in the art can easily understand how to determine the identity of two polypeptides or two nucleic acids.For example, identity can be calculated after aligning the two sequences so that identity is at its highest level.

[0135] Alternative methods for calculating identity can be performed using published algorithms. Optimal alignment of sequences for comparison may be performed using the local identity algorithm of Smith and Waterman, Adv. Appl. Math 2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection.

[0136] The same type of identity can be obtained for nucleic acids by algorithms disclosed in, for example, Zuker, Science 244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA 86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306 (1989), which are incorporated herein by reference at least for material related to nucleic acid alignment. While it is understood that either method can typically be used and that in some cases the results of these various methods may differ, one skilled in the art will understand that if identity is found by at least one of these methods, the sequence is said to have the described identity and be disclosed herein.

[0137] Protein modifications include modifications of the amino acid sequence. Modifications in the amino acid sequence can occur naturally as allelic variations (e.g., due to genetic polymorphisms), can arise due to environmental influences (e.g., exposure to ultraviolet light), or can be created by human intervention (e.g., by mutagenesis of cloned DNA sequences), such as induced point mutations, deletion mutations, insertion mutations, and substitution mutations. These modifications can result in changes in 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 types: substitution, insertion, or deletion. Insertions include amino- and / or carboxyl-terminal fusions and intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than insertions of amino- or carboxyl-terminal fusions, e.g., about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2 to 6 residues are deleted at any one site within the protein molecule. Amino acid substitutions are typically single residue substitutions but can occur at several different positions at a time; insertions usually involve about 1-10 amino acid residues, and deletions range from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., a deletion of two residues or an insertion of two residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. Mutations should not place the sequence out of reading frame and preferably do not create complementary regions that could generate secondary mRNA structure. Substitutional modifications involve the removal of at least one residue and the insertion of a different residue in its place. Such substitutions are generally made according to Table 2 below and are referred to as conservative substitutions. Table 2. Exemplary amino acid substitutions [Table 2-1] [Table 2-2]

[0138] Modifications, including specific amino acid substitutions, are made by known methods. For example, modifications can be made by site-directed mutagenesis of nucleotides in the DNA encoding the polypeptide, thereby generating DNA encoding the modifications, followed by expression of the DNA in recombinant cell culture. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis.

[0139] Modifications can be selected to optimize binding. For example, affinity maturation techniques can be used to alter the binding of scFvs by introducing random mutations into the complementarity determining regions (CDRs). Such random mutations can be introduced using a variety of techniques, including radiation, chemical mutagens, and error-prone PCR. Multiple rounds of mutation and selection can be performed, for example, using phage display.

[0140] The present disclosure also relates to nucleic acids encoding the chimeric polypeptides described herein, and the use of such nucleic acids to produce the chimeric polypeptides and for therapeutic purposes. For example, the present invention includes DNA and RNA molecules (e.g., mRNA, self-replicating RNA) that encode the chimeric polypeptides, and therapeutic uses of such DNA and RNA molecules.

[0141] Exemplary Compositions Exemplary fusion proteins of the invention combine the above elements in various orientations. The orientations described in this section are meant as example orientations and should not be considered limiting.

[0142] In some embodiments, the fusion protein comprises an IL-12 polypeptide, a blocking moiety, and a half-life extending element. In some embodiments, the IL-12 polypeptide is positioned between the half-life extending element and the blocking moiety. In some embodiments, the IL-12 polypeptide is N-terminal to the blocking moiety and the half-life extending element. In some such embodiments, the IL-12 polypeptide is proximal to the blocking moiety, and in some such embodiments, the IL-12 polypeptide is proximal to the half-life extending element. At least one protease-cleavable linker must be included in all embodiments so that the IL-12 polypeptide can be active upon cleavage. In some embodiments, the IL-12 polypeptide is C-terminal to the blocking moiety and the half-life extending element. The additional elements may be linked to each other by a cleavable linker, a non-cleavable linker, or by direct fusion.

[0143] In some embodiments, the blocking domain used is capable of extending half-life, and the IL-12 polypeptide is positioned between two such blocking domains, hi some embodiments, the IL-12 polypeptide is positioned between two blocking domains, one of which is capable of extending half-life.

[0144] In some embodiments, the two cytokines are contained in the same construct. In some embodiments, each cytokine has two blocking domains attached (for a total of three in one molecule), with one blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extending domains may be included to optimize pharmacokinetic properties.

[0145] In some embodiments, the three cytokines are contained in the same construct, and in some embodiments, the third cytokine may function to block the other two cytokines in place of the blocking domain between the two cytokines.

[0146] Preferred half-life extending elements for use in the fusion proteins are human serum albumin (HSA), an antibody or antibody fragment (e.g., scFV, dAb) that binds serum albumin, human or humanized IgG, or a fragment of any of the foregoing. In some preferred embodiments, the blocking moiety is human serum albumin (HSA), or an antibody or antibody fragment that binds serum albumin, an antibody that binds to a cytokine and prevents binding or activation of a cytokine receptor, another cytokine, or a fragment of any of the foregoing. In preferred embodiments including an additional targeting domain, the targeting domain is an antibody that binds to a cell surface protein that is enriched on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin.

[0147] Therapeutic methods and pharmaceutical compositions Additionally provided are methods for treating a subject having or at risk of developing a disease or disorder, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. The method comprises administering to a subject in need thereof an effective amount of a fusion protein disclosed herein, typically administered as a pharmaceutical composition. In some embodiments, the method further comprises selecting a subject having or at risk of developing such a disease or disorder. The pharmaceutical composition preferably comprises a blocked IL-12 polypeptide, fragment, or mutein thereof that is activated at sites of inflammation. In one embodiment, the chimeric polypeptide comprises an IL-12 polypeptide, fragment, or mutein thereof, and a serum half-life extending element. In another embodiment, the chimeric polypeptide comprises an IL-12 polypeptide, fragment, or mutein thereof, and a blocking moiety, e.g., a steric-blocking polypeptide, which can sterically block the activity of the IL-12 polypeptide, fragment, or mutein. In another embodiment, the chimeric polypeptide comprises an IL-12 polypeptide, a fragment or mutein thereof, a blocking moiety, and a serum half-life extending element.

[0148] Inflammation is part of the complex biological response of body tissues to harmful stimuli, such as pathogens, damaged cells, and irritants. It is a defensive reaction involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cellular injury, remove necrotic cells and tissue damaged by the original injury and inflammatory process, and initiate tissue repair. Inflammation can occur due to infection, as a symptom, or as a disease, such as cancer, atherosclerosis, allergies, myopathy, HIV, obesity, or autoimmune disease. Autoimmune diseases are chronic conditions resulting from an abnormal immune response to self-antigens. Autoimmune diseases that can be treated with the polypeptides disclosed herein include, but are not limited to, lupus, celiac disease, type 1 diabetes mellitus, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, and systemic lupus erythematosus.

[0149] The pharmaceutical composition can include one or more protease-cleavable linker sequences. The linker sequence serves to provide flexibility between polypeptides so that each polypeptide can inhibit the activity of the first polypeptide. The linker sequence can be positioned between any or all of the cytokine polypeptide, its fragment or mutein, the blocking moiety, and the serum half-life extending element. Optionally, the composition includes two, three, four, or five linker sequences. The linker sequence, the two, three, or four linker sequences can be the same or different. In one embodiment, the linker sequence includes GGGGS (SEQ ID NO: 87), GSGSGS (SEQ ID NO: 88), or G(SGGG)2SGGT (SEQ ID NO: 89). In another embodiment, the linker includes a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 24), GPLGVRG (SEQ ID NO: 83), IPVSLRSG (SEQ ID NO: 84), VPLSLYSG (SEQ ID NO: 85), and SGESPAYYTA (SEQ ID NO: 86).

[0150] Suitable linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, 2 to 15 amino acids, 3 to 12 amino acids, including 4 to 10 amino acids, 1 to 9 amino acids, 6 to 8 amino acids, or 7 to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0151] In some embodiments, the linker is cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, elastase, PR-3, granzyme M, calpain, matrix metalloproteinase (MMP), plasminogen activator, cathepsin, caspase, tryptase, or tumor cell surface protease.

[0152] Further provided are methods for treating a subject having or at risk of developing cancer. The methods include administering to a subject in need thereof an effective amount of a chimeric polypeptide (fusion protein) disclosed herein, typically administered as a pharmaceutical composition. In some embodiments, the methods further include selecting a subject having or at risk of developing cancer. The pharmaceutical composition preferably comprises a blocked cytokine, a fragment thereof, or a mutant protein thereof that is activated at the tumor site. Preferably, the tumor is a solid tumor. The cancer may be colon cancer, lung cancer, melanoma, sarcoma, renal cell carcinoma, or breast cancer.

[0153] The method may further include administering one or more additional agents to treat the cancer, such as a chemotherapeutic agent (e.g., Adriamycin, Cervidine, Bleomycin, Alkeran, Velban, Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene, Noantrone, Thiguanine, Cytaribine, Procarabizine), an immunotherapy agent (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), a cellular therapy agent (e.g., CAR-T, T-cell therapy), an oncolytic virus, etc.

[0154] Provided herein are 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 refers to a material that is not biologically or otherwise undesirable, i.e., such a material may be administered to a subject without causing undesired biological effects or interacting in a deleterious manner with other components of the pharmaceutical formulation or composition in which it is contained. The carrier is selected to minimize degradation of the active ingredient and to minimize adverse side effects in the subject.

[0155] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 stEdition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to render the formulation isotonic; however, if desired, the formulation can be hypertonic or hypotonic. Examples of pharmacologically acceptable carriers include, but are not limited to, sterile water, saline, buffers such as Ringer's solution, and dextrose solution. The pH of the solution is generally about 5 to about 8 or about 7 to 7.5. Other carriers include sustained-release preparations, such as semipermeable matrices of solid hydrophobic polymers containing the immunogenic polypeptide. The matrices are in the form of shaped articles, such as films, liposomes, or microparticles. Certain carriers may be more preferable depending, for example, on the route of administration and the concentration of the composition being administered. The carrier is suitable for administration of the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide to humans or other subjects.

[0156] Pharmaceutical formulations or compositions are administered in several ways, depending on whether local or systemic treatment is desired and on the area to be treated. The compositions may be administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intraarticular, intraperitoneal, intramuscular, subcutaneous, intracavity, transdermal, intrahepatic, intracranial, including nebulization / inhalation, or by placement via bronchoscopy. In some embodiments, the compositions are administered locally (non-systemically), such as intratumorally, intraarticularly, or intrathecally.

[0157] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives are optionally present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like.

[0158] Formulations for topical administration include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like are optionally necessary or desirable.

[0159] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tables. Thickeners, flavorings, diluents, emulsifiers, dispersing aids, or binders are optionally desirable.

[0160] Optionally, the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide is administered via a vector. Several compositions and methods are available for delivering nucleic acid molecules and / or polypeptides to cells in vitro or in vivo, e.g., via expression vectors. These methods and compositions can be broadly divided into two categories: viral and non-viral delivery systems. Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, e.g., to produce cell lines that express and preferably secrete the encoded chimeric polypeptide, or to therapeutically deliver nucleic acids to a subject. The components of the chimeric nucleic acids disclosed herein are typically operably linked in-frame to encode a fusion protein.

[0161] As used herein, a plasmid or viral vector is a substance that transports the disclosed nucleic acid into a cell without degradation and contains a promoter that drives expression of the nucleic acid molecule and / or polypeptide in the cell. Viral vectors include, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, Sindbis, and other RNA viruses, including those with an HIV backbone. Also preferred are any virus families that share the properties of these viruses and are suitable for use as vectors. Retroviral vectors are reviewed by Coffin et al., Retroviruses, Cold Spring Harbor Laboratory Press (1997), which is incorporated herein by reference for information on vectors and methods for their production. The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83 (1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques 15:868-72 (1993)). The advantage and use of these viruses as vectors is that they can replicate within the initially infected cell but are unable to form new infectious viral particles, limiting the extent to which they can spread to other cell types. Recombinant adenoviruses have been shown to achieve high efficiency following direct in vivo delivery to respiratory epithelia, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replicating vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors.

[0162] The provided polypeptides and / or nucleic acid molecules can be delivered via virus-like particles. Virus-like particles (VLPs) are composed of viral protein(s) derived from viral structural proteins. Methods for producing and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0163] The provided polypeptides can be delivered by subviral dense bodies (DBs), which transport proteins to target cells by membrane fusion. Methods for making and using DBs are described, for example, in Pepperl-Klindworth et al., Gene Therapy 10:278-84 (2003).

[0164] The provided polypeptides can be delivered by exoskeleton aggregates. Methods for making and using exoskeleton aggregates are described in International Publication No. WO2006 / 110728.

[0165] Non-viral delivery methods can include expression vectors containing a nucleic acid molecule and a nucleic acid sequence encoding the polypeptide, where the nucleic acid is operably linked to an expression control sequence. Suitable vector backbones include those routinely used in the art, such as plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce chimeric polypeptides by expression in appropriate host cells, such as CHO cells.

[0166] Preferred promoters for controlling transcription from vectors in mammalian host cells may be derived from a variety of sources, such as from the genomes of viruses such as polyoma virus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, and most preferably, cytomegalovirus (CMV), or from heterologous mammalian promoters, such as the β-actin promoter or EF1α promoter, or from hybrid or chimeric promoters (e.g., the CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0167] Enhancers generally refer to DNA sequences that function at variable distances from the transcription start site and can be 5' or 3' to the transcription unit. Furthermore, enhancers can be located within introns or within the coding sequence itself. They are typically 10 to 300 base pairs (bp) in length and function in cis. Enhancers typically function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate transcriptional regulation. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), and enhancers from eukaryotic viruses are typically used for general expression. Preferred examples include the SV40 enhancer on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

[0168] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated by environmental factors such as temperature and light. Optionally, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In certain vectors, the promoter and / or enhancer region may be active in a cell-type-specific manner. Optionally, in certain vectors, the promoter and / or enhancer region may be active in all eukaryotic cells, regardless of cell type. Preferred promoters of this type are the CMV promoter, the SV40 promoter, the β-actin promoter, the EF1α promoter, and retroviral long terminal repeats (LTRs).

[0169] Vectors can also include, for example, an origin of replication and / or a marker. Marker genes can confer a selectable phenotype, such as antibiotic resistance, to cells. The marker product is used to determine whether the vector has been delivered to a cell and, once delivered, whether it is expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into mammalian host cells, the transformed mammalian host cells can survive when placed under selective pressure. Other examples of markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Additionally, expression vectors can include tag sequences designed to facilitate 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™ tags (Kodak; New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0170] As used herein, the terms peptide, polypeptide, or protein are used broadly to refer to two or more amino acids linked by a peptide bond. Protein, peptide, and polypeptide are also used interchangeably herein to refer to an amino acid sequence. It should be recognized that the term polypeptide is not used herein to imply a specific size or number of amino acids comprising a molecule; the peptides of the present invention can contain up to a few or more amino acid residues. As used throughout, a subject can be a vertebrate, more specifically, a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. Such terms do not denote a particular age or sex. Thus, both adult and newborn subjects, regardless of gender, are intended to be encompassed. As used herein, patient or subject may be used interchangeably and may refer to a subject with a disease or disorder (e.g., cancer). The terms patient or subject include human subjects and veterinary subjects.

[0171] A subject at risk of developing a disease or disorder may have a genetic predisposition to the disease or disorder, e.g., have a family history of the disease, or have a genetic mutation that causes the disease or disorder, or exhibit early signs or symptoms of the disease or disorder. A subject who currently has a disease or disorder may have one or more symptoms of the disease or disorder and may have been diagnosed with the disease or disorder.

[0172] The methods and medicaments described herein are useful for both preventive and therapeutic treatments. For preventive use, a therapeutically effective amount of a chimeric polypeptide or a chimeric nucleic acid sequence encoding a chimeric polypeptide described herein is administered to a subject pre-onset (e.g., before overt signs of cancer or inflammation) or during early onset (e.g., at the time of early signs and symptoms of cancer or inflammation). Prophylactic administration can occur from 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. Therapeutic treatment involves administering a therapeutically effective amount of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide described herein to a subject after the diagnosis or onset of cancer or inflammation (e.g., an autoimmune disease). Prophylactic use can also be applied when a patient is undergoing a treatment in which inflammation is expected, such as chemotherapy.

[0173] According to the methods taught herein, a subject is administered an effective amount of an agent (e.g., a chimeric polypeptide). The terms effective amount and effective dosage are used interchangeably. The term effective amount is defined as any amount necessary to produce a desired physiological response. Effective amounts and schedules for administering an agent may be determined empirically, and making such determinations is within the skill of one of ordinary skill in the art. The dosage range for administration is one large enough to produce the desired effect, in which one or more symptoms of a disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects, such as undesirable cross-reactions, anaphylactic reactions, and the like. Generally, dosages 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 can be determined by one of ordinary skill in the art. Dosages can be adjusted by the individual physician if there are any contraindications. Dosages can vary, and one or more doses can be administered daily, for one day or for several days. Guidance can be found in the literature as to appropriate dosages for given classes of pharmaceuticals.

[0174] As used herein, the terms treatment, treating, or treating refer to a method of reducing the effects of a disease or condition or the symptoms of that disease or condition. Thus, in the disclosed methods, treatment can refer to 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 therapeutic if there is a 10% reduction in one or more symptoms of the disease in a subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to native or control levels. It is understood that treatment does not necessarily refer to a cure or complete elimination of the disease, condition, or the symptoms of the disease or condition.

[0175] As used herein, the terms prevent, preventing, and prevention of a disease or disorder refer to an action, such as administering a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, before or at about the same time that a subject begins to exhibit one or more symptoms of the disease or disorder, that inhibits or delays the onset or progression of one or more symptoms of the disease or disorder. As used herein, reference to decrease, reduction, or inhibition includes a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more change compared to control levels. Such terms can include, but do not necessarily include, complete elimination.

[0176] Disclosed are materials, compositions, and components that can be used in, combined with, used in preparation for, or are products of the disclosed methods and compositions. These and other materials are disclosed herein, and when combinations, subsets, interactions, groups, etc. of these materials are disclosed, it is understood that although specific reference to each of the various individual and aggregate combinations and permutations of these compounds may not be expressly disclosed, each is specifically contemplated and described herein. For example, when a method is disclosed and discussed, and several modifications that can be made to several molecules comprising that method are discussed, all combinations and permutations of that method, and possible modifications, are specifically contemplated unless clearly indicated otherwise. Likewise, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including, but not limited to, steps in methods using the disclosed compositions. Thus, if there are a variety of additional steps that may be performed, it is understood that each of these additional steps may be performed with any specific method step or combination of method steps of the disclosed methods, and that each such combination or subset of combinations is to be considered specifically contemplated and disclosed.

[0177] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. [Example]

[0178] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced in light of the general description provided herein.

[0179] Example 1: Protease cleavage of fusion proteins by MMP9 protease Those skilled in the art will be familiar with how to set 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 number SAE0078-50 or Enzo catalog number BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein can then be used in functional analysis or stored at -80°C before testing. The extent of cleavage was monitored by SDS PAGE using methods well known in the art.

[0180] Example 2: HEK Blue assay HEK-Blue IL12 cells (InvivoGen) were seeded at 250,000 cells / well in suspension in medium with or without 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL12, chimeric IL12 (mouse p35 / human p40), or activatable hIL12 for 24 hours at 37°C and 5% CO2. The activity of uncleaved and cleaved activatable hIL12 was tested. Inducible cleaved hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen), a colorimetric assay.

[0181] Example 3: Splenocyte T-Blast assay T-blasts were induced from mouse splenocytes by incubation with PHA for 6 days and recombinant hIL12 for 24 hours. Tblasts were then seeded at 200,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA) and stimulated with serial dilutions of recombinant hIL12, chimeric IL12 (mouse p35 / human p40), or mouse IL12 for 72 hours at 37°C, 5% CO2. The activity of uncleaved and cleaved forms of IL12 was tested. Inducible cleaved hIL12 was generated by incubation with active MMP9. IL12 activity was assessed by downstream quantification of IFNγ production using the mIFNγ alphaLISA.

[0182] Example 4: In vivo delivery of protease-activated fusion proteins results in reduced tumor growth The chimeric polypeptides will be examined to determine whether they have potential biological effects in vivo. These experiments use a system in which intraperitoneally injected tumor cells rapidly and preferentially bind and grow primarily in milky spots, a series of organized immune aggregates found in the omentum (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system allows for multiple intraperitoneal delivery of fusion proteins and allows tumor growth to be analyzed by examining dissociated omental cells, providing a convenient method for examining the effects of fusion protein treatment on tumor growth. 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 low levels of MMP2 and MMP9, but MMP levels increase when Colon38 tumors are present in the omentum. This tumor model will be used to examine the ability of IL-2 mutein fusion proteins to affect tumor growth. Colon38 cells are injected intraperitoneally and allowed to attach and grow for 1 day before being treated daily with the fusion protein intraperitoneally. On day 7, animals are sacrificed and examined for tumor growth using flow cytometry and colony formation assays.

[0183] Example 5: Construction of an exemplary activatable interleukin protein that targets CD20 Generation of activatable interleukin domains The human IL-12p35 chain reference sequence is Uniprot accession number P29459. The human IL-12p40 chain reference sequence is Uniprot accession number P29460. IL-12p35 and IL-12p40 are cloned into an expression construct. A protease cleavage site is introduced between the IL-12p35 and IL-12p40 domains. An IL-12 polypeptide capable of binding to a CD20 polypeptide present in a tumor or on a tumor cell is produced as follows: A nucleic acid is generated to contain (1) a nucleic acid sequence encoding an IFNg polypeptide sequence and (2) one or more polypeptide linker nucleic acid sequences. The activatable interleukin plasmid construct, which can optionally contain a Flag, His, or other affinity tag, is electroporated into HEK293 or other suitable human or mammalian cell line and purified. Validation assays include T cell activation assays using T cells that are responsive to IL-12 stimulation in the presence of a protease.

[0184] Generation of scFv CD20-binding domains CD20 is a cell surface protein present on B lymphocytes. The CD20 antigen is found on normal and malignant pre-B lymphocytes and mature B lymphocytes, including those found in over 90% of B-cell non-Hodgkin's lymphomas (NHL). The antigen is not found on hematopoietic stem cells, activated B lymphocytes (plasma cells), or normal tissues. Therefore, several antibodies, primarily of murine origin, namely 1F5, 2B8 / C2B8, 2H7, and 1H4, have been described.

[0185] Thus, human or humanized anti-CD20 antibodies are used to generate scFv sequences 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 constructs' codons are optionally optimized for expression in Homo sapiens cells. The order of the VL and VH domains in the scFv is changed (i.e., VL-VH or VH-VL orientation), and three copies of the subunit "G4S" (SEQ ID NO: 87) or (G4S)3 (SEQ ID NO: 90) link the variable domains to create the scFv domain. The anti-CD20 scFv plasmid constructs, which can optionally contain Flag, His, or other affinity tags, are electroporated into HEK293 or other appropriate human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20-expressing cells.

[0186] Cloning of DNA expression constructs encoding activatable interleukin proteins. An activatable interleukin protein is constructed by combining an anti-CD20 scFv domain and a serum half-life extender (e.g., an HSA-binding peptide or a VH domain) using an activatable interleukin construct with a protease cleavage site domain. For expression of the activatable interleukin protein in CHO cells, the coding sequences for all protein domains are cloned into a mammalian expression vector system. Briefly, gene sequences encoding the activatable interleukin domain, serum half-life extender, and CD20-binding domain are separately synthesized and subcloned with peptide linkers L1 and L2. The resulting constructs are then linked together in the following order to obtain the final construct: CD20-binding domain-L1-IL-12p35-L2-protease cleavage domain-L3-IL-12p40-L4-anti-CD20 scFv-L5-serum half-life extender. All expression constructs are designed to contain coding sequences for an N-terminal signal peptide and a C-terminal hexahistidine (6xHis) tag (SEQ ID NO: 91) to facilitate protein secretion and purification, respectively.

[0187] Expression of activatable interleukin proteins in stably transfected CHO cells The CHO cell expression system (Flp-In®, Life Technologies), derived from CHO-K1 Chinese hamster ovary cells (ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4):1275-81), is used. Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0188] To adapt cells to growth in suspension, they are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium containing 10% DMSO.

[0189] Recombinant CHO cell lines stably expressing secreted, activatable interleukin proteins are generated by transfection of suspension-adapted cells. Upon selection with the antibiotic hygromycin B, viable cell density is measured twice weekly, and cells are centrifuged to a maximum density of 0.1 × 10 6 Resuspend in fresh selection medium at 1000 viable cells / mL. After 2-3 weeks of selection, harvest cell pools stably expressing the activatable interleukin protein, at which point transfer the cells to standard medium in shake flasks. Perform protein gel electrophoresis or flow cytometry to confirm expression of the recombinant secreted protein. Cryopreserve stable cell pools in DMSO-containing medium.

[0190] Activatable interleukin proteins are produced by secretion into cell culture supernatant in stably transfected CHO cell lines in fed-batch cultures for 10 days. After 10 days, cell culture supernatant is typically harvested at >75% culture viability. Samples are taken from the production cultures every other day to assess cell density and viability. On the day of harvest, the cell culture supernatant is removed by centrifugation and vacuum filtered before further use.

[0191] Protein expression levels and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

[0192] Purification of activatable interleukin proteins Activatable interleukin proteins are purified from CHO cell culture supernatants in a two-step procedure. In the first step, the constructs are subjected to affinity chromatography, followed by preparative size-exclusion chromatography (SEC) on Superdex 200 in the second step. The samples are buffer-exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg / mL. The purity and homogeneity of the final samples (usually >90%) are assessed by SDS-PAGE under reducing and non-reducing conditions, followed by immunoblotting using anti-HSA or anti-idiotypic antibodies, and analytical SEC, respectively. The purified proteins are stored in equal aliquots at -80°C until use.

[0193] Example 6: Determination of antigen affinity by flow cytometry The activatable interleukin proteins of the preceding examples were prepared using human CD20 + Cells and cynomolgus monkey CD20 + Their binding affinity to cells is tested.

[0194] 100 μL of serial dilutions of the activatable interleukin protein of the preceding example and CD20 with at least one protease + After washing three times with FACS buffer, the cells were incubated with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotypic antibody in the same buffer for 45 minutes on ice. After a second washing cycle, the cells were incubated with 0.1 mL of 15 μg / mL FITC-conjugated goat anti-mouse IgG antibody under the same conditions as before. As a control, the cells were incubated with anti-His IgG without activatable interleukin protein, followed by incubation with FITC-conjugated goat anti-mouse IgG antibody. The cells were then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 x 10 4The fluorescence of live cells is measured using a Beckman-Coulter FC500 MPL flow cytometer (Beckman-Coulter, Krefeld, Germany) using MXP software or a Millipore Guava EasyCyte flow cytometer (Merck Millipore, Schwalbach, Germany) using Incyte software. The mean fluorescence intensity of the cell samples is calculated using CXP software (Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck Millipore, Schwalbach, Germany). After subtracting the fluorescence intensity values ​​of cells stained with the secondary and tertiary reagents alone, the values ​​are then calculated using GraphPad Prism (version 6.00 for Windows, GraphPad Software, La Jolla, CA). K using the equation for single-site binding (hyperbolic) D Used to calculate the value.

[0195] CD20 binding and cross-reactivity were evaluated using human CD20 + Evaluate with tumor cell lines. K D The ratios were calculated using the K determined for CHO cell lines expressing recombinant human or recombinant cynomolgus antigens. D Calculate using the value.

[0196] Example 7: Cytotoxicity Assay The activatable interleukin protein of the preceding example was prepared by the addition of its CD20 + Mediating an immune response against target cells is assessed in vitro.

[0197] Fluorescently labeled CD20 +REC-1 cells (mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with PBMCs isolated from random donors or CB15 T cells (normalized T cell line) as effector cells in the presence of the activatable interleukin protein of the preceding example and at least one protease. After 4 hours of incubation at 37°C in a humidified incubator, the release of fluorescent dye from the target cells into the supernatant is measured using a spectrofluorometer. Target cells incubated without the activatable interleukin protein of the preceding example and target cells completely lysed by the addition of saponin at the end of the incubation serve as negative and positive controls, respectively.

[0198] Based on the measured remaining live target cells, calculate the percentage of specific cell lysis according to the following formula: [1-(live target cells)] (試料) Number of living targets (自然発生的) ) × 100%. Sigmoidal dose-response curves and EC 50 Calculate values. The lysis values ​​obtained for a given antibody concentration are used to calculate a sigmoidal dose-response curve by a four-parameter logistic fit analysis using Prism software.

[0199] Example 8: Pharmacokinetics of activatable interleukin proteins The activatable interleukin proteins of the preceding examples are evaluated in animal studies for half-time loss.

[0200] The activatable interleukin protein is administered to cynomolgus monkeys as a 0.5 mg / kg bolus injection into the saphenous vein. Another group of cynomolgus monkeys receives a cytokine of comparable size but lacking a serum half-life extending element. A third and fourth group receive a cytokine with a serum half-life extending element and a cytokine with CD20 and a serum half-life extending element, respectively, both of comparable size to the activatable interleukin protein. Each test group consists of five monkeys. Serum samples are collected at the indicated time points and serially diluted, and protein concentrations are determined using a binding ELISA against CD20.

[0201] Pharmacokinetic analysis is performed using test article plasma concentrations. Group mean plasma data for each test article follow a multiexponential profile when plotted against time post-dose. Data are fit with a standard two-compartment model using the bolus dose and first-order rate constants for the distribution and elimination phases. The general equation for best fit of intravenous data is c(t) = Ae -αt +Be -βt where c(t) is the plasma concentration at time t, A and B are the intercepts on the Y-axis, and α and β are the apparent first-order rate constants for the distribution and elimination phases, respectively. The α phase is the initial phase of clearance and reflects the distribution of the protein to all extracellular fluids of the animal, while the second or β 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), B = D / V(β-k21) / (α-β), where α and β (if α > β) are fitted to a quadratic equation, r 2 +(k12+k21+k10)r+k21k10=0, with the estimated parameters V=volume of distribution, k10=elimination rate, k12=rate of transfer from compartment 1 to compartment 2, k21=rate of transfer from compartment 2 to compartment 1, and D=administered dose.

[0202] Data Analysis: Concentration versus time profile graphs are generated using KaleidaGraph (KaleidaGraph™ V.3.09 Copyright 1986-1997. Synergy Software. Reading, Pa.). Values ​​reported as below the measurement sensitivity (LTR) are not included in the PK analysis and are not plotted. Pharmacokinetic parameters are determined by compartmental analysis using WinNonlin software (WinNonlin® Professional V.3.1 WinNonlin™ Copyright 1998-1999. Pharsight Corporation. Mountain View, Calif.). Pharmacokinetic parameters are calculated as described in Ritschel WA and Kearns GL, 1999, IN: Handbook of Basic Pharmacokinetics Including Clinical Applications, 5th edition, American Pharmaceutical Assoc., Washington, DC.

[0203] The activatable interleukin proteins of the preceding examples are expected to have improved pharmacokinetic parameters, such as increased elimination half-times, compared to proteins lacking the serum half-life extending element.

[0204] Example 9: Xenograft tumor model The activatable interleukin proteins of the preceding examples are evaluated in xenograft models.

[0205] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and 4 × 10 6 Ramos RA1 cells were inoculated subcutaneously into the right dorsal flank. Tumors were 100–200 mm 3 Once the animals reach 100 mg / kg, they are assigned to three treatment groups: 1.5 x 10 mg / kg in Group 2 and 3 (8 animals each); 7of activated human T cells are injected intraperitoneally. Three days later, animals in group 3 are subsequently treated with 50 μg of the activatable interleukin protein of the preceding example for a total of nine intravenous doses (once daily for nine days). Groups 1 and 2 are treated with vehicle only. Body weights and tumor volumes are measured for 30 days.

[0206] Animals treated with the activatable interleukin proteins of the preceding examples are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0207] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of these claims and their equivalents be covered thereby. Example 10: MC38 Experiment We used the MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, to investigate the ability of the fusion protein to affect tumor growth. Example 10a: MC38 IFNa and IL-12 Drugs and Treatments: [Table 4] procedure: Mice were anesthetized with isoflurane for transplantation of ulcer-reducing cells. CR female C57BL / 6 mice were injected with 5 × 10 cells subcutaneously in the flank. 5 The tumors were prepared with 0% Matrigel containing MC38 tumor cells. The cell injection volume was 0.1 mL per mouse. Mice were 8-12 weeks old on the day of initiation. The tumors had an average size of 100-150 mm. 3Once the weight loss reached 10%, pair-matching was performed and treatment was initiated. Body weights were measured at baseline and then twice weekly until termination. Caliper measurements were taken twice weekly until termination. Any adverse reactions were reported immediately. Individual animals with a single observed weight loss of more than 30% or three consecutive measurements of more than 25% weight loss 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. Individuals within groups with a weight loss of more than 20% who reached an individual weight loss endpoint were euthanized. If the group treatment associated with weight loss recovered to within 10% of its original weight, dosing was resumed at a lower dose or less frequent dosing schedule. Exceptions to the non-treated % weight recovery were allowed on a case-by-case basis. The endpoint was tumor growth delay (TGD). Animals were monitored individually. The experimental endpoint was a tumor volume of 1500 mm 3 or 45 days, whichever came first. Responders were followed thereafter. When the endpoint was reached, the animals were euthanized.

[0208] Example 11: Conditionally active fusion proteins containing a blocking moiety that is a serum albumin binding domain This example describes the production and activity of a fusion protein, preferably a cytokine, that has inducible activity, i.e., is inactive until induced, typically by separation of the blocking moiety from the active moiety upon cleavage of a linker between the blocking moiety and the active moiety. The fusion protein contains a single antibody variable domain (dAb) that binds serum albumin through a CDR loop and is linked to an active moiety (here, an anti-CD3 scFV) through one or more non-CDR loops (e.g., C-loops). The serum albumin-binding blocking moiety is operably linked to the active moiety via a protease-cleavable linker, and the active moiety is operably linked to a targeting domain (here, an anti-epidermal growth factor receptor (EGFR) dAb or an anti-prostate-specific membrane antigen (PSMA) dAb) via a linker that is not protease-cleavable. These fusion proteins can be administered as inactive proteins that are activated upon cleavage of the protease-cleavable linker and subsequent release of the inhibitory albumin-binding domain. The anti-CD3 scFV in the fusion protein is a substitute for the desired cytokine in the fusion proteins described in this disclosure. Similar fusion proteins containing a desired cytokine (e.g., IL-2, IL-12, interferon) or a functional fragment or mutein thereof, a targeting domain, and an albumin-binding dAb that also binds to and inhibits the cytokine or its functional fragment or mutein can be prepared using the methods described and exemplified herein. An anti-serum albumin dAb that binds to a desired cytokine or its functional fragment or mutein and inhibits its activity can provide both steric masking of the cytokine (due to the proximity of the cytokine to the bound serum albumin) and specific masking of the cytokine (due to binding to the cytokine via a non-CDR loop (e.g., C-loop)). Anti-serum albumin dAbs that bind to a desired cytokine or its functional fragment or mutein and inhibit its activity can be obtained using appropriate methods, such as by introducing amino acid sequence diversity into the non-CDR loop (e.g., C-loop) of an anti-serum albumin-binding dAb and screening for binding to the desired cytokine.Any suitable method can be used for selection, such as phage display. For example, an exemplary anti-serum albumin dab that can be used has the following sequence: The amino acid sequence within the C-loop (bold and underlined) can be diversified (e.g., randomized) and the resulting dAbs screened for binding to serum albumin via CDR interactions and for binding to cytokines via non-CDR loop interactions. If desired, the amino acid sequence of a known cytokine-binding peptide can be introduced into the C-loop. [ka]

[0209] A. Protease activation of ProTriTAC leads to a significant increase in in vitro activity Purified ProTriTAC (prodrug), non-cleavable ProTriTAC [prodrug (non-cleavable)], and recombinant active drug fragments mimicking 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 potency in a T cell-dependent cytotoxicity assay.

[0210] For ELISA, soluble ProTriTAC protein at an appropriate concentration was incubated with immobilized recombinant human CD3e (R&D Systems) in PBS supplemented with 15 mg / ml human serum albumin for 1 h at room temperature. Plates were blocked using SuperBlock (Thermo Fisher), washed with PBS containing 0.05% Tween-20, and detected using the noncompetitive anti-CD3 idiotypic monoclonal antibody 11D3, followed by peroxidase-conjugated secondary antibody and TMB-ELISA substrate solution (Thermo Fisher).

[0211] For flow cytometry, the indicated concentrations of soluble ProTriTAC protein were incubated with purified human primary T cells in the presence of 2% fetal bovine serum in PBS and 15 mg / ml human serum albumin for 1 hour at 4°C. Plates were washed with 2% fetal bovine serum in PBS and detected with the AlexaFluor 647-labeled noncompetitive anti-CD3 idiotypic monoclonal antibody 11D3. Data were analyzed using FlowJo 10 (FlowJo, LLC).

[0212] For functional potency in a T cell-dependent cytotoxicity assay, the indicated concentrations of soluble ProTriTAC protein were incubated with purified resting human T cells (effector cells) and HCT116 cancer cells (target cells) at an effector:target cell ratio of 10:1 for 48 hours at 37° C. The HCT116 target cell line was stably transfected with a luciferase reporter gene, allowing measurement of specific T cell-mediated cell killing by ONE-Glo (Promega).

[0213] B. ProTriTAC exhibits potent protease-dependent antitumor activity in rodent tumor xenograft models ProTriTAC was evaluated for its in vivo antitumor activity in HCT116 subcutaneous xenograft tumors mixed with expanded human T cells in immunocompromised NCG mice. Specifically, on day 0, 5 x 10 HCT116 cells were mixed with 2.5 x 10 expanded T cells per mouse. ProTriTAC administration began the following day and was administered intraperitoneally on a once-daily schedule of 10 x 10. At the indicated times, tumor volume measurements were obtained using caliper measurements and calculated using the formula V = (length x width x width) / 2.

[0214] C. Expression, purification and stability of exemplary ProTriTAC trispecific 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 6x histidine tag (SEQ ID NO: 91). Expi293F cells (Life Technologies A14527) were maintained in suspension in Optimum Growth Flasks (Thomson) at 0.2–8x1e6 cells / ml in Expi293 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 stable pools, which were then cultured in production medium for up to 12 days before purification. The amount of exemplary fusion protein in the conditioned medium was quantified using an Octet RED 96 instrument (ForteBio / Pall) with a Protein A chip, using a control fusion protein for a standard curve. Conditioned medium from either host cell was filtered and partially purified by affinity and desalting chromatography. The fusion protein was then polished by ion exchange and formulated into a neutral buffer containing additives along with the fraction pool. Final purity was assessed using SDS-PAGE and analytical SEC using an Acquity BEH SEC 200 1.7u 4.6 x 150mm column (Waters Corporation), split into aqueous / organic mobile phases with neutral pH additives on a 1290 LC System, and peak integration using Chemstation CDS software (Agilent). The fusion protein purified from CHO host cells is shown in the SDS-PAGE below.

[0215] Stability assessment The purified fusion protein in the two formulations was further divided equally into sterile tubes and stressed by five freeze-thaw cycles, each consisting of >1 hour at -80°C and room temperature, or by incubation at 37°C for 1 week. Stressed samples were assessed for concentration and turbidity by UV spectrometry using UV-transparent 96-well plates (Corning 3635) with SpectraMax M2 and SoftMaxPro software (Molecular Devices), SDS-PAGE, and analytical SEC, and compared to the same analyses for unstressed control samples. An overlay of analytical SEC chromatograms of control and stressed samples for a single exemplary ProTriTAC molecule purified from 293 host cells is shown below.

[0216] The results show that ProTriTAC was produced in yields comparable to regular TriTAC from CHO stable pools, and that the protein was stable after repeated freeze-thaw cycles and after 1 week at 37°C.

[0217] D. Demonstration of Functional Masking and Stability of ProTriTAC In Vivo in a 3-Week Cynomolgus Monkey Pharmacokinetic Study Single doses of PSMA-directed ProTriTAC (SEQ ID NO: 74), uncleavable ProTriTAC (SEQ ID NO: 75), unmasked / uncleavable TriTAC (SEQ ID NO: 78), and protease-activated ProTriTAC (SEQ ID NO: 76) mimicking the active drug were administered to cynomolgus monkeys at 0.1 mg / kg by intravenous injection. Plasma samples were collected at the indicated time points. Concentrations of ProTriTAC were measured using biotinylated recombinant human PSMA (R&D The pharmacokinetic parameters were determined using ligand binding assays with Sigma-Aldrich systems and sulfo-tagged anti-CD3 idiotypic antibody cloned 11D3 in an MSD assay (Meso Scale Diagnostic, LLC). Pharmacokinetic parameters were estimated using Phoenix WinNonlin pharmacokinetic software using noncompartmental methods consistent with the intravenous bolus administration route.

[0218] To calculate the in vivo prodrug conversion rate, the circulating blood concentration of the active drug is calculated using the following simultaneous differential equations: [where P is the concentration of the prodrug, A is the concentration of the active drug, and k a is the rate of prodrug activation in the circulation, and k c,P is the clearance rate of the prodrug, and k c,A is the clearance rate of the active drug].

number

[0219] Prodrug, active drug, and non-cleavable prodrug control (k c,NCLV The clearance rates of cleavable and non-cleavable prodrugs were empirically determined in cynomolgus monkeys. To estimate the rate of prodrug activation in the circulation, it was assumed that the difference in clearance rate between cleavable and non-cleavable prodrugs arises solely from non-specific activation in the circulation. Therefore, the rate at which a prodrug is converted to an active drug in the circulation was estimated by subtracting the clearance rate of the cleavable prodrug from the clearance rate of the non-cleavable prodrug.

number

[0220] The initial concentration of circulating prodrug was determined empirically, and the initial concentration of active drug was assumed to be zero.

[0221] Results and Discussion The results of this Example 11 demonstrate that fusion proteins containing a polypeptide with a desired therapeutic activity, such as a cytokine or functional fragment or mutein thereof, or an anti-CD3 scFV, can be prepared such that the therapeutic activity is masked by a masking domain that binds both serum albumin and the active polypeptide. The masking domain is operably linked to the active domain via a protease-cleavable linker. The results demonstrate that fusion proteins of this type can be administered as inactive proteins that are activated upon protease cleavage at the desired site of therapeutic activity, such as a tumor.

[0222] The amino acid sequences of the fusion proteins used in Example 11 are shown as SEQ ID NOs: 71 to 78.

[0223] Exemplary fusion protein constructs are detailed in Table 3. 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] Sequence Listing [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8] [Table 5-9] [Table 5-10] [Table 5-11] [Table 5-12] [Table 5-13] [Table 5-14] Incorporation by Reference

[0224] The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entirety for all purposes.

[0225] Other embodiments The above disclosure may encompass multiple separate inventions with independent utility. While each of these inventions has been disclosed in its preferred form(s), numerous variations are possible, and the specific embodiments thereof disclosed and exemplified herein should not be considered limiting. The subject matter of this disclosure includes all novel and unobvious combinations and combinations of the various elements, features, functions, and / or properties disclosed herein. The following claims particularly point out certain combinations and combinations that are deemed novel and unobvious. Inventions embodied in other combinations and combinations of features, functions, elements, and / or properties may be claimed in this application, in applications claiming priority from this application, or in related applications. Such claims, whether directed to different inventions or the same invention, and whether broader, narrower, equal, or different in scope than the original claims, are also deemed to be within the inventive subject matter of this disclosure. The present invention provides, for example, the following items. (Item 1) formula [A]-[L1]-[D] or [A]-[L1]-[D]-[L2]-[B] or [B]-[L1]-[A]-[L1]-[D] [In the formula, A is an interleukin-12 (IL-12) polypeptide; B is a half-life extending element; L1 and L2 are each independently a polypeptide linker, wherein L1 is a protease-cleavable polypeptide linker and L2 is an optionally protease-cleavable polypeptide linker; D is an IL-12 blocking moiety; The fusion polypeptide is an attenuated IL-12 receptor agonist, but upon cleavage of (i) the L1 protease-cleavable polypeptide linker, or (ii) when L2 is a protease-cleavable polypeptide linker, upon cleavage of both L1 and L2, the IL-12 polypeptide-containing fragment of the fusion polypeptide produced is an IL-12 receptor agonist with potency and half-life equivalent to that of a naturally occurring IL-12 polypeptide. (Item 2) a) interleukin-12 (IL-12) polypeptide [A]; b) an IL-12 blocking moiety [D], and c) a protease-cleavable polypeptide linker [L] a fusion polypeptide comprising at least one of each of: The fusion polypeptide, wherein the IL-12 polypeptide and the IL-12 blocking moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated IL-12 receptor activating activity, wherein the IL-12 receptor activating activity of the fusion polypeptide is at least about 10-fold less than the IL-12 receptor activating activity of a polypeptide containing the IL-12 polypeptide produced by cleavage of the protease-cleavable polypeptide linker. (Item 3) 3. The fusion polypeptide according to item 2, wherein [A] is the N-terminus of the fusion polypeptide. (Item 4) [A] is the formula [A1]-[L3]-[A2] or [A2]-[L3]-[A1], wherein A1 is an IL-12p40 subunit polypeptide; A2 is an IL-12p35 subunit polypeptide; 4. The fusion polypeptide of any one of items 1 to 3, wherein L3 is an optionally protease-cleavable polypeptide linker. (Item 5) 5. The fusion polypeptide according to item 4, wherein [A] has the formula [A1]-[L3]-[A2], and L3 is not protease cleavable. (Item 6) 6. The fusion polypeptide of any one of items 1 to 5, wherein the agonistic activity of the IL-12 polypeptide containing the fragment of the truncated polypeptide is increased by at least 10-fold compared to the agonistic activity of the uncleaved fusion polypeptide. (Item 7) 7. The fusion polypeptide of item 6, wherein the agonist activity is assessed using a HEK Blue reporter cell assay. (Item 8) 8. The fusion polypeptide of any one of items 1 to 7, wherein each protease-cleavable polypeptide linker independently comprises a sequence capable of cleavage 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 activation protein (FAP), ADAM metalloproteinase, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease. (Item 9) 9. The fusion polypeptide of item 8, 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 10) 10. The fusion polypeptide of any one of items 1 to 9, wherein the IL-12 blocking moiety inhibits activation of the IL-12 receptor by the fusion polypeptide. (Item 11) 11. The fusion polypeptide of any one of items 1 to 10, wherein the IL-12 blocking moiety is non-covalently bound to the IL-12 polypeptide. (Item 12) 12. The fusion polypeptide of any one of items 1 to 11, wherein the IL-12 blocking moiety binds to the IL-12 p40 subunit polypeptide. (Item 13) 12. The fusion polypeptide of any one of items 1 to 11, wherein the IL-12 blocking moiety binds to the IL-12 p35 subunit polypeptide. (Item 14) 12. The fusion polypeptide of any one of items 1 to 11, wherein the IL-12 blocking moiety binds to IL-12 p70. (Item 15) 15. The fusion polypeptide of any one of items 1 to 14, wherein the IL-12 blocking moiety comprises a ligand-binding domain or fragment of a cognate receptor for IL-12, a single domain antibody, Fab or scFv that binds to the IL-12 polypeptide, or an antibody or antibody fragment selected from a single domain antibody, Fab, and scFv that binds to the receptor for IL-12. (Item 16) 16. The fusion polypeptide of any one of items 1 to 15, wherein the IL-12 blocking moiety is also a half-life extending element. (Item 17) 17. The fusion polypeptide of any one of items 1 to 10 or 16, wherein the IL-12 blocking moiety sterically blocks the agonist activity of the IL-12. (Item 18) 18. The fusion polypeptide of item 17, wherein the IL-12 blocking moiety is human serum albumin or an antigen-binding polypeptide that recruits human serum albumin. (Item 19) 19. The fusion polypeptide of any one of items 1 to 18, wherein the IL-12 is freely dissociated from the IL-12 blocking moiety after the protease-cleavable polypeptide linker is cleaved by a protease. (Item 20) 20. The fusion polypeptide according to any one of items 1 to 19, wherein the fusion polypeptide binds to the IL-2 receptor. (Item 21) 21. The fusion polypeptide of any one of items 1 to 20, further comprising at least one half-life extending element. (Item 22) 22. The fusion polypeptide of item 21, wherein the half-life extending element is human serum albumin or an antigen-binding polypeptide that binds to human serum albumin. (Item 23) 22. The fusion polypeptide of item 21, wherein the half-life extending element is an immunoglobulin Fc. (Item 24) 12. The fusion polypeptide of item 11, wherein the non-covalent association is pH dependent. (Item 25) 25. The fusion polypeptide of any one of items 1 to 24, wherein IL-12 receptor activation is determined using a standard in vitro receptor activation assay and equal amounts of the IL-12 polypeptide and the fusion polypeptide on a molar basis. (Item 26) 26. The fusion polypeptide of any one of items 1 to 25, wherein IL-12 is freely dissociated from the IL-12 blocking moiety and / or half-life extending element after the protease-cleavable sequence is cleaved by a protease. (Item 27) Further comprising a half-life extending element [B], [B]-[L1]-[A]-[L2]-[D] 24. The fusion polypeptide according to any one of items 2 to 23, comprising: wherein L1 and L2 are each independently a polypeptide linker, L1 is a protease-cleavable polypeptide linker, and L2 is an optionally protease-cleavable polypeptide linker. (Item 28) 28. The fusion polypeptide of item 27, wherein L2 is a protease-cleavable linker. (Item 29) 29. The fusion polypeptide of any one of items 1 or 3 to 28, wherein L1 is a substrate for a first protease and L2 is a substrate for a second protease. (Item 30) 30. The fusion polypeptide according to any one of items 1 to 29, further comprising a tumor-specific antigen-binding peptide. (Item 31) 31. The fusion polypeptide of claim 30, wherein the tumor-specific antigen-binding peptide is linked to the IL-12 polypeptide by a non-cleavable linker. (Item 32) 31. The fusion polypeptide of claim 30, wherein the tumor-specific antigen-binding peptide is linked to the IL-12 polypeptide, the half-life extending element, or the IL-12 blocking moiety by a cleavable linker. (Item 33) 33. The fusion polypeptide of any one of items 1 to 32, further comprising a linker between the domains of IL-12. (Item 34) 33. The fusion polypeptide of any one of Aspects 3 to 32, wherein the serum half-life of the IL-12 polypeptide-containing fragment produced by protease cleavage of the protease-cleavable polypeptide linker is equivalent to the half-life of naturally occurring IL-12. (Item 35) A nucleic acid encoding the polypeptide according to any one of items 1 to 34. (Item 36) 36. A vector comprising the nucleic acid of item 35. (Item 37) A host cell containing the vector according to item 36. (Item 38) A method for producing a pharmaceutical composition, comprising culturing the host cell according to Item 37 under conditions suitable for expression and harvesting of a desired polypeptide. (Item 39) A pharmaceutical composition comprising i) an effective amount of the fusion polypeptide according to any one of items 1 to 34, and ii) a pharmacologically acceptable additive. (Item 40) 35. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of the fusion polypeptide of any one of items 1 to 34. (Item 41) 35. The fusion polypeptide according to any one of items 1 to 34 for use as a medicament. (Item 42) 35. The fusion polypeptide of any one of items 1 to 34 for use in treating a tumor in a subject in need thereof. (Item 43) 35. A pharmaceutical composition for treating a tumor in a subject in need thereof, comprising, as an active ingredient, the fusion polypeptide according to any one of items 1 to 34.

Claims

[Claim 1] The invention as described in the drawings of this application.

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