Activatable cytokine polypeptides and use methods thereof

Abstract

To provide activatable cytokine polypeptides and use methods thereof.SOLUTION: The disclosure features a fusion protein that is a conditionally active variant of a cytokine of interest. In one aspect, the full-length polypeptide of the invention has reduced or minimal cytokine-receptor activating activity even though it contains a functional cytokine polypeptide. Upon activation, e.g., by cleavage of a linker that joins a blocking moiety, e.g. a steric blocking polypeptide, in sequence to the active cytokine, the cytokine can bind its receptor and effect signaling. Typically, the fusion protein further comprises an in vivo half-life extension element, which may be cleaved from the cytokine in the tumor microenvironment.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, U.S. Provisional Application No. 62 / 756,515, filed November 6, 2018, and U.S. Provisional Application No. 62 / 756,507, 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 105365-0020_SL.txt and is 866,384 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 undesirable self-reactive 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 important aspects of mature peripheral Treg and antigen-activated conventional T cells. IL-2 has been extensively studied due to its potent T cell growth factor activity in vitro, in part because this activity has provided a powerful means to directly promote immunity in patients with, e.g., cancer and AIDS-HIV, or a target to antagonize undesirable responses, e.g., transplant rejection and autoimmune diseases. In vitro studies with IL-2 provided a strong rationale for these studies, but 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 some cytokines is well known and described, and some cytokines are already 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, multiple myeloma, etc. 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 some types of tumors, and therefore are the subject of further research. 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 mutants of a cytokine of interest. In one aspect, the full-length polypeptides of the present invention have reduced or minimal cytokine receptor activation activity even though they contain a functional cytokine polypeptide. Upon activation, such as by cleavage of a linker that in turn connects a blocking moiety, such as a steric blocking polypeptide, to an active cytokine, the cytokine, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, IFN alpha, IFN beta, IFN gamma, TNF alpha, lymphotoxin, TGF-β1, TGF beta 2, TGF beta 3, GM-CSF, CXCL10, CCL19, CCL20, CCL21, or a functional fragment or mutant protein of any of the foregoing, can bind to its receptor and carry out 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 may also contain a blocking polypeptide moiety that also extends serum half-life and / or directs the full-length polypeptide to the desired site of cytokine activity. Alternatively, the full-length fusion polypeptide may contain a serum half-life extension element and / or a targeting domain that is separate from the blocking polypeptide moiety. Preferably, the fusion protein contains at least one element or domain that can extend circulating half-life in vivo. Preferably, this element is enzymatically removed at the desired site in the body (e.g., cleaved by proteases in the tumor microenvironment), restoring substantially similar pharmacokinetic properties to the payload molecule (e.g., IL2 or IFNa) as the naturally occurring payload molecule. The fusion protein may be 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 may be attached to the cytokine via a cleavable or non-cleavable linker.When linked by a non-cleavable linker, the targeting domain may further help to retain the cytokine in the tumor and may be considered as a retention domain.The targeting domain does not necessarily have to be directly linked to the payload molecule, but may be directly linked to another element of the fusion protein.This is especially true when the targeting domain is linked by a cleavable linker.

[0007] In one aspect, a fusion polypeptide is provided that includes a cytokine polypeptide, or a functional fragment or mutein thereof, and a blocking moiety, such as a steric blocking domain. The blocking moiety can be fused to the cytokine polypeptide directly or via a linker and separated from the cytokine 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 cytokine polypeptide is fused to the blocking moiety via a linker that contains a protease cleavage site, the cytokine polypeptide can be released from the blocking moiety upon protease-mediated cleavage of the linker and bind to its receptor. The linker is designed to be cleaved at the site of desired cytokine activity, such as 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 directly binds to FcRn or binds to human serum albumin.

[0009] In addition to the serum half-life extending element, the pharmaceutical composition described herein preferably comprises 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 construct of the present invention is cleaved at a protease cleavage site, for example, in a disease-specific microenvironment of a subject or in blood, and that the targeting domain(s) bind to the target antigen on a target cell. 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, an injured red blood cell, an arterial plaque cell, or a fibrous tissue cell.

[0011] The target antigen is sometimes expressed on the surface of diseased cells or tissues, such as tumor or cancer cells. Target antigens of tumors include, but are not limited to, fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, FAP, and CEA. The pharmaceutical compositions disclosed herein also include proteins that include 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 polypeptides specifically bind to a cell surface molecule. In some embodiments, the targeting polypeptides specifically bind to a tumor antigen. In some embodiments, the targeting polypeptides specifically and independently bind 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 polypeptides specifically and independently bind to two different antigens, at least one of the antigens being a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the targeting polypeptides function as retention domains and are attached to the cytokine via a non-cleavable linker.

[0013] As described herein, a cytokine blocking moiety is capable of binding to a cytokine and 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 the conditionally active proteins, vectors or host cells containing such nucleic acids, and pharmaceutical compositions to 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, a graft-versus-host disease or a 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, a graft-versus-host disease or a 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, a graft-versus-host disease or a host-versus-graft disease. [Brief description of the drawings]

[0019] [Figure 1-1]Schematic diagram showing a cytokine or chemokine containing a blocking moiety that is activated by a protease. 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, thus 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] Schematic diagram showing a protease-activated cytokine or chemokine, where HSA (blocking moiety) is directly linked to the cytokine or chemokine of interest, and a protease cleavage site is between HSA and the cytokine or chemokine of interest. Left of the arrow, the cytokine is connected to the blocking moiety via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. Right of the arrow, in an inflammatory or tumor environment, proteases cleave at the protease cleavage site on the linker, releasing the blocking moiety and allowing the cytokine to bind to its receptor. [Figure 1-3] 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, thus 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 the receptor. The cytokine now has similar pK properties (e.g., shorter half-life) compared to the native cytokine. [Figure 1-4] 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 a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or 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. [Diagram 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 a 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 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, thus liberating the serum half-life extending element and the blocking moiety, allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties (e.g., shorter half-life) compared to the native cytokine. [Diagram 3]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 the targeting domain via a protease-cleavable linker, thus blocking the cytokine's ability to bind to its receptor. To the right of the arrow, in an inflammatory or tumor microenvironment, a protease cleaves at the protease cleavage site in the linker, liberating the targeting domain and the blocking moiety, allowing the cytokine to bind to its receptor. [Figure 4-1] 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 a protease-cleavable linker. The left side of the arrow shows that the cytokine polypeptide is connected to the targeting domain, the blocking moiety, and the half-life extending element via the protease-cleavable linker(s), thereby blocking the ability of the cytokine polypeptide 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) to release the half-life extending element, the targeting domain, and the blocking moiety, allowing the cytokine to bind to its receptor. The cytokine here has similar pK properties (e.g., shorter half-life) compared to the native cytokine. [Figure 4-2]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. The left side of the arrow shows that the cytokine is connected to the targeting domain, the blocking moiety, and the half-life extending element via the protease-cleavable linker(s), thus 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(s) to release the half-life extending element and the blocking moiety, allowing the cytokine to bind to the receptor. The targeting moiety remains attached, maintaining the cytokine in the tumor microenvironment. The cytokine here has similar pK properties (e.g., shorter half-life) compared to the native cytokine. [Diagram 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 the 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-2. In this example, the binding site for serum albumin can be formed by the CDRs of the serum albumin binding domain. [Figure 7-1] 7a-7h are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo® (Promega), a luminescence-based cell viability assay. Each proliferation assay was performed with (FIGS. 7b, 7d, 7f, 7h) and without (FIGS. 7a, 7c, 7e, 7g) HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 7-2] 7a-7h are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo® (Promega), a luminescence-based cell viability assay. Each proliferation assay was performed with (FIGS. 7b, 7d, 7f, 7h) and without (FIGS. 7a, 7c, 7e, 7g) HSA. Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease-cleaved forms of the fusion protein were used in each assay. [Figure 8] 8a-8f are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo (Promega), a luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 9-1]Figures 9a-z are a series of graphs showing the activity of exemplary IL-2 fusion proteins in the IL-2-dependent cytotoxic T lymphocyte cell line CTLL-2. Each graph shows the results of an IL-2 proliferation assay quantified by CellTiter-Glo (Promega), a luminescence-based cell viability assay. Both uncleaved and MMP9 protease-cleaved forms of the fusion proteins were used in each assay. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above. [Figure 9-5] Same as above. [Figure 9-6] Same as above. [Figure 10] The results of a protein cleavage assay are shown. The fusion protein ACP16 was run on an SDS-PAGE gel in both cleaved and uncleaved forms. As shown in the gel, cleavage was complete. [Figure 11] Figures 11a and 11b are graphs showing the results from a HEK-Blue IL-12 reporter assay performed on human p40 / mouse p35 IL12 fusion protein before and after protease cleavage. The analysis 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 12-1] Figures 12a-f show a series of graphs showing the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that activity is greater in the truncated IL12 than in the full fusion protein. The constructs tested were ACP06 (Figure 12a), ACP07 (Figure 12c), ACP08 (Figure 12b), ACP09 (Figure 12d), ACP10 (Figure 12e), and ACP11 (Figure 12f). [Figure 12-2]Figures 12a-f show a series of graphs showing the results of HEK-blue assays of four IL-12 fusion proteins before and after cleavage by MMP9. The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). The data show that activity is greater in the truncated IL12 than in the full fusion protein. The constructs tested were ACP06 (Figure 12a), ACP07 (Figure 12c), ACP08 (Figure 12b), ACP09 (Figure 12d), ACP10 (Figure 12e), and ACP11 (Figure 12f). [Figure 13] The results of a protein cleavage assay are shown. 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 14] FIG. 1 is a schematic diagram showing a non-limiting example of an inducible cytokine protein, the construct being activated upon cleavage by a protease of the linker attached between the two subunits of the cytokine. [Figure 15] Figures 15a-d are graphs showing 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 16] 16a-f are a series of graphs depicting the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using a WEHI279 cell viability assay. Each assay was performed using media containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease cleaved forms of the fusion protein were used in each assay. [Figure 17]17a-f are a series of graphs showing the activity of exemplary IFNγ fusion proteins compared to the activity of a mouse IFNγ control using a B16 reporter assay. Each assay was performed using media containing HSA (+HSA) or not containing HSA (-HSA). Each fusion protein contained an anti-HSA conjugate, and both uncleaved and MMP9 protease cleaved forms of the fusion protein were used in each assay. [Figure 18] Figures 18a and 18b show the results of the protein cleavage assay described in Example 2. Two constructs, ACP31 (an IFN-a fusion protein; Figure 18a) and ACP55 (an IFN-g fusion protein; Figure 18b), were run on an SDS-PAGE gel in both cleaved and uncleaved forms. As shown in the gel, cleavage was complete. [Figure 19] 19a and 19b are a series of graphs showing the activity of exemplary IFNγ fusion proteins before and after protease cleavage using a B16 reporter assay (FIGS. 19a and 19b). Each assay was performed with medium containing HSA, and each fusion protein contains an anti-HSA conjugate. Both uncleaved and MMP9 protease cleaved forms of the fusion proteins were used in each assay. [Figure 20] Figures 20a and 20b are a series of graphs showing the activity of exemplary IFNa fusion proteins before and after cleavage using a B16 reporter assay (20a and 20b). Each assay was performed with medium containing HSA, and each fusion protein contains an anti-HSA conjugate. Both uncleaved and MMP9 protease cleaved forms of the fusion protein were used in each assay. [Figure 21] Figures 21a-d are a series of graphs showing the results of a tumor growth study using the MC38 cell line. Figures 21a-c show the effect on tumor growth of IFNγ and IFNγ fusion proteins injected intraperitoneally (IP) using different dose levels and schedules (ug = micrograms, BID = twice daily, BIW = twice weekly, QW = once weekly). Figure 21d shows the effect on tumor growth of intratumoral (IT) injection of IFNγ and IL-2. [Figure 22] Figures 22a and 22b are a series of graphs showing the activity of exemplary MMP9 protease cleaved IFNγ fusion proteins (ACP51 and ACP52) compared to the activity of the uncleaved fusion proteins using a B16 reporter assay, where each fusion protein contains an anti-HSA binding domain and a tumor tropic domain. [Diagram 23] Figures 23a and 23b are a series of graphs showing the activity of exemplary MMP9 protease cleaved IFNγ fusion proteins (ACP53 and ACP54) compared to the activity of the uncleaved fusion proteins using a B16 reporter assay. Each fusion protein contains IFNγ fused directly to albumin. [Figure 24-1] Figures 24a-d are graphs showing results obtained from a HEK-Blue IL-2 reporter assay performed with IL-2 fusion proteins and recombinant human IL2 (Rec hIL-2). The analysis was based on quantification of secreted alkaline phosphatase (SEAP) activity using the reagent QUANTI-Blue (InvivoGen). [Figure 24-2] Same as above. [Figure 24-3] Same as above. [Figure 24-4] Same as above. [Figure 25-1]Figures 25a and 25b are two graphs showing the analysis of ACP16 (Figure 25a) and ACP124 (Figure 25b) in a HEKBlue IL-2 reporter assay in the presence of HSA. Circles indicate the activity of the uncleaved polypeptide and squares indicate the activity of the cleaved polypeptide. Figure 25c is a graph showing the results of a CTLL-2 proliferation assay. CTLL2 cells (ATCC) were suspension seeded at a concentration of 500,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA) and stimulated with a dilution series of activatable hIL2 for 72 hours at 37°C, 5% CO2. The activity of uncleaved and cleaved forms of activatable ACP16 was tested. The cleaved forms of activatable hIL2, generated by incubation with active MMP9, were used to assess cell activity using the luminescence-based cell viability assay CellTiter-Glo (Promega). The circles represent the intact fusion protein and the boxes represent the protease-cleaved fusion protein. [Figure 25-2] Same as above. [Figure 26-1] Figure 26 is a series of graphs showing the activity of fusion proteins in a HEKBlue IL-12 reporter assay. Figure 26a shows IL-12 / STAT4 activation in comparison with ACP11 (human p40 / mouse p35 IL12 fusion protein) and ACP04 (negative control). [Figure 26-2] Figure 26b is a series of graphs showing the activity of fusion proteins in a HEKBlue IL-12 reporter assay. Figure 26b is a graph showing the analysis of ACP91 (a chimeric IL-12 fusion protein). The squares show the activity of the uncleaved ACP91 polypeptide, and the triangles show the activity of the cleaved polypeptide (ACP91 + MMP9). The EC50 values ​​for each are shown in the table. [Figure 26-3] Figure 26c is a series of graphs showing the activity of fusion proteins in the HEKBlue IL-12 reporter assay. Figure 26c is a graph showing the analysis of ACP136 (a chimeric IL-12 fusion protein). The squares show the activity of the uncleaved ACP136 polypeptide, and the triangles show the activity of the cleaved polypeptide (ACP136+MMP9). The EC50 values ​​for each are shown in the inset table. [Figure 27-1] Figures 27a-f are a series of graphs showing that the truncated IFNα1 polypeptides ACP31 (Figure 27a), ACP125 (Figure 27b), and ACP126 (Figure 27c) are active in the HEKBlue reporter assay. The EC50 values ​​for each IFNα construct are shown in the table below each graph. [Figure 27-2] Same as above. [Figure 27-3] Same as above. [Figure 28-1] Figures 28a-n are a series of graphs showing the activity of APC56 (Figure 28a), APC57 (Figure 28b), APC58 (Figure 28c), APC59 (Figure 28d), APC60 (Figure 28e), APC61+HSA (Figure 28f), ACP30+HSA (Figure 28g), ACP73 (Figure 28h), ACP70+HSA (Figure 28i), ACP71 (Figure 28j), ACO72 (Figure 28k), ACP73 (Figure 28l), ACP74 (Figure 28m), and ACP75 (Figure 28n) in a B16 IFNα reporter assay. Each fusion was tested for its activity when cleaved (squares) and uncleaved (circles). Analysis of mouse IFNγ was included in each graph as a comparative control. [Figure 28-2] Same as above. [Figure 28-3] Same as above. [Figure 28-4] Same as above. [Figure 28-5] Same as above. [Figure 28-6] Same as above. [Figure 28-7] Same as above. [Figure 28-8] Same as above. [Figure 28-9] Same as above. [Figure 28-10] Same as above. [Figure 28-11] Same as above. [Figure 28-12] Same as above. [Figure 29-1]29A-29B are graphs showing the results of analyzing ACP31 (mouse IFNα1 fusion protein) in tumor xenograft models. FIG. 29A shows tumor volume over time in mice treated with 33 μg ACP31 (circles), 110 μg ACP31 (triangles), 330 μg ACP31 (diamonds), and 1 μg mouse wild-type IFNa1 (dashed line, squares) and 10 μg mIFNa1 (dashed line, small circles) as controls. Vehicle alone is shown as large open circles. The data show that tumor volume is reduced over time in mice treated with ACP31 in a dose-dependent manner. [Figure 29-2] FIG. 29b is a graph showing the results of analyzing ACP11 (human p40 / mouse p35 IL12 fusion protein) in a tumor xenograft model. FIG. 29b shows tumor volume over time in mice treated with 17.5 μg ACP11 (squares), 175 μg ACP31 (triangles), 525 μg ACP31 (circles), and 2 μg ACP04 (dashed line, triangles) and 10 μg ACP04 (dashed line, diamonds) as controls. Vehicle alone is shown as a large open circle. The data show that tumor volume is reduced over time in both ACP11 and ACP04 (human p40 / mouse p35 IL12 fusion protein) treated mice in a dose-dependent manner. [Diagram 30] Figures 30a-30f are a series of spaghetti plots showing tumor volume over time in a mouse xenograft tumor model for mice treated with vehicle alone (Figure 30a), 2 μg ACP04 (Figure 30b), 10 μg ACP04 (Figure 30c), 17.5 μg ACP11 (Figure 30d), 175 μg ACP11 (Figure 30e), and 525 μg ACP11 (Figure 30f). Each line represents one mouse. [Figure 31-1]31A-31D are graphs showing the results of analyzing ACP16 in tumor xenograft models. FIG. 31A shows tumor volume over time in mice treated with 4.4 μg ACP16 (squares), 17 μg ACP16 (triangles), 70 μg ACP16 (inverted triangles), 232 μg ACP16 (filled circles), and as comparative controls, 12 μg wild-type IL-2 (dashed line, triangles) and 36 μg wild-type IL-2 (dashed line, diamonds). Vehicle alone is shown as a large open circle. The data show that tumor volume is reduced over time in a dose-dependent manner in mice treated with high concentrations of ACP16. [Figure 31-2] 31b is a graph showing the results of analyzing ACP124 in a tumor xenograft model. Figure 31b shows tumor volume over time in mice treated with 17 μg ACP124 (squares), 70 μg ACP124 (triangles), 230 μg ACP124 (inverted triangles), and 700 μg ACP124. Vehicle alone is shown as a large open circle. [Figure 31-3] 1 is a graph showing the analysis of ACP16 and ACP124 in a tumor xenograft model. Tumor volume over time is shown in mice treated with 17 μg ACP16 (triangles), 70 μg ACP16 (circles), 232 μg ACP16 (filled circles), and controls 17 μg ACP124 (dashed line, triangles), 70 μg ACP124 (dashed line, diamonds), and 230 μg ACP124 (dashed line, diamonds). Vehicle alone is shown as inverted filled triangles. Data show a dose-dependent decrease in tumor volume over time in mice treated with ACP16, but not ACP124. [Figure 32-1] Figures 32a-c are a series of spaghetti plots showing the activity of fusion proteins in the MC38 mouse xenograft model, corresponding to the data shown in Figure 31. Each line in the plots is one mouse. [Figure 32-2] Same as above. [Figure 32-3] Same as above. [Diagram 33] FIG. 1 is a graph showing tumor volume over time in a mouse xenograft model showing tumor growth in control mice (open circles) and AP16-treated mice (squares). [Figure 34-1]Figure 34 is a series of survival plots showing survival of mice over time following treatment with cleavable fusion proteins: Figure 34a shows data for mice treated with vehicle alone (grey line), 17 μg ACP16 (dark line), and 1 μg ACP124 (dashed line). [Figure 34-2] Figure 34b is a series of survival plots showing survival of mice over time following treatment with cleavable fusion proteins. Figure 34b shows data for mice treated with vehicle alone (grey line), 70 μg ACP16 (dark line), and 70 μg ACP124 (dashed line). [Figure 34-3] Figure 34c is a series of survival plots showing survival of mice over time following treatment with cleavable fusion proteins. Figure 34c shows data for mice treated with vehicle alone (grey line), 232 μg ACP16 (dark line), and 230 μg ACP124 (dashed line). [Figure 34-4] Figure 34d shows a series of survival plots showing survival of mice over time following treatment with cleavable fusion proteins. Figure 34d shows data for mice treated with vehicle alone (grey line), 232 μg ACP16 (dark line), and 700 μg ACP124 (dashed line). [Diagram 35] FIG. 1 is a series of spaghetti plots showing the activity of fusion proteins in an MC38 mouse xenograft model. All groups of mice received a total of four doses, except for the three highest doses of APC132, where lethal toxicity was detected after 1 week / twice dosing. Shown are vehicle alone (top row), 17 μg, 55 μg, 70 μg, and 230 μg of ACP16 (all top rows), 9 μg, 28 μg, 36 μg, and 119 μg of ACP132 (all middle rows), and 13 μg, 42 μg, 54 μg, and 177 μg of ACP21 (all bottom rows). Each line in the plot represents an individual animal. [Diagram 36] 1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. [Figure 37] 1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. [Figure 38]1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. [Figure 39] 1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. [Diagram 40] 1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. [Diagram 41] 1 shows the properties of the TriTAC polypeptide used as an example of a protease-cleavable fusion protein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Disclosed herein are methods and compositions for engineering and using constructs containing inducible cytokines. Cytokines are potent immune agonists, which has led them to be considered as promising therapeutic agents in oncology. However, cytokines have proven to have a very narrow therapeutic window. They have short serum half-lives and are considered to be 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 the biology of cytokines and the inability to effectively target and control their activity, cytokines have not achieved the expected clinical benefits in the treatment of tumors.

[0021] Disclosed herein are fusion proteins that overcome the problems of toxicity and short half-life that have severely limited the clinical use of cytokines in oncology. The fusion proteins contain cytokine polypeptides with receptor agonist activity. However, in the context of the fusion proteins, the cytokine receptor agonist activity is attenuated and the circulating half-life is extended. The fusion proteins contain protease cleavage sites that are cleaved by proteases associated with the desired site of cytokine activity (e.g., tumor) and are typically concentrated or preferentially present at the site of desired activity. Thus, the fusion proteins preferentially (or selectively) and efficiently cleave at the desired site of activity to substantially restrict cytokine activity to the desired site of activity, such as the tumor microenvironment. Cleavage by proteases at the desired site of activity, such as within the tumor microenvironment, liberates a form of cytokine from the fusion protein that is much more active as a cytokine receptor agonist than the fusion protein (typically at least about 100 times more active than the fusion protein). The form of cytokine released upon cleavage of the fusion protein typically has a short half-life, often substantially similar to the half-life of naturally occurring cytokine, further restricting cytokine activity to the tumor microenvironment.Although the half-life of the fusion protein is extended, the circulating fusion protein is attenuated, and toxicity is dramatically reduced or eliminated, since active cytokine is directed to the tumor microenvironment.The fusion protein described herein allows for the first time to administer an effective therapeutic amount of cytokine to treat tumors, which substantially restricts the activity of cytokine to the tumor microenvironment, dramatically reducing or eliminating the undesirable systemic effects and toxicity of cytokine.

[0022] Unless otherwise defined, all technical terms, notations, and other scientific terms used herein are intended to have the meaning commonly understood by those of ordinary skill in the art to which the present invention pertains. In some cases, terms with commonly understood meanings are defined herein for ease of understanding and / or ready reference, and the inclusion of such definitions herein should not necessarily be construed as representing a departure from what is commonly understood in the art. The techniques and procedures described or referenced herein are generally well understood and may be easily understood by those of ordinary skill in the art using conventional methodology, e.g., Sambrook et al. Generally, molecular cloning methods are used, such as those described in widely used molecular cloning methodologies such as those described in, for example, W. et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are generally performed according to manufacturer-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 (such as 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, IL-5 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] Cytokines are well known to have a short half-life in serum, often only a few minutes or hours. Even cytokine forms with a modified amino acid sequence intended to extend the half-life in serum but retaining receptor agonist activity typically have a short half-life in serum. As used herein, "cytokine with a short half-life" refers to a cytokine that has a substantially short time for its half-life to circulate in the serum of a subject, for example, a cytokine with a serum half-life of less than 10 minutes, less than 15 minutes, less than 30 minutes, less than 60 minutes, less than 90 minutes, less than 120 minutes, less than 240 minutes, or less than 480 minutes. As used herein, cytokines with a short half-life include cytokines whose sequences have not been modified to achieve a longer half-life than normal in the body of a subject, and polypeptides with a modified amino acid sequence intended to extend the serum half-life but retaining receptor agonist activity. In the latter case, it is not intended to include the addition of heterologous protein domains, such as authentic half-life extension elements, such as serum albumin, etc.

[0025] "Sortase" is a peptide transferase that modifies a protein by recognizing and cleaving a carboxyl-terminal localization signal embedded in or bound to the target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (X is any standard amino acid) between the Thr residue and the Gly residue on the target protein, and the Thr residue transiently binds to the Cys residue, which is the active site on the enzyme, to form an enzyme-thioacyl intermediate. To complete peptide transfer and create a peptide-monomer complex, a biomolecule with an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displaces sortase A, and links the two molecules.

[0026] As used herein, the term "steric blocker" refers to a polypeptide or polypeptide moiety that can be covalently bound directly or indirectly to a cytokine polypeptide, for example in the form of a chimeric polypeptide (fusion protein) or the like, through other moieties such as linkers, but is not otherwise covalently bound to the cytokine polypeptide. A steric blocker can be non-covalently bound to a cytokine polypeptide, for example through electrostatic, hydrophobic, ionic or hydrogen bonds. A steric blocker typically inhibits or blocks the activity of a cytokine moiety by virtue of its proximity to and comparable size to the cytokine moiety. A steric blocker may also block by recruiting large protein binding partners. An example of this is an antibody that binds to serum albumin; the antibody itself may or may not be large enough to block activation or binding alone, but recruitment of albumin allows sufficient steric blocking.

[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 the disclosed nucleic acid to a cell in an undegraded form and contains a promoter that effects expression of the nucleic acid molecule and / or polypeptide in the delivered cell.

[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 that make up the molecule, and that 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., 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 either sex 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 a symptom of the 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 a symptom of the 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 a disease, condition, or a symptom 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 administration of 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 an appropriate control level. Such terms can include, but do not necessarily include, the complete loss of a function or property, such as, for example, agonist activity.

[0036] An "attenuated cytokine receptor agonist" is a cytokine receptor agonist that has reduced receptor agonist activity compared to a naturally occurring agonist of 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 of that receptor. When a fusion protein containing a cytokine polypeptide as described herein is described as "attenuated" or has "attenuated 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 cleavage with a protease. Although a domain may be removable by cleavage with a protease 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 the molecule, and that function can be performed by the moiety in the context of another molecule. A moiety may be a chemical 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 may be a protein domain, such as serum albumin. Blocking may be achieved by steric blocking substances or specific blocking substances. Steric blocking substances block by size and position, not based on specific binding, an example being serum albumin. Specific blocking substances block by specific interactions with the portion to be blocked. Specific blocking substances need to be tailored to the specific cytokine or active domain, and steric blocking substances can be used regardless of the payload as long as it is large enough.

[0039] In general, the therapeutic use of cytokines is strongly limited by their systemic toxicity. For example, TNF was initially discovered for its ability to induce hemorrhagic necrosis of some tumors and for its in vitro cytotoxic effects on various tumor lines, but has since been shown to have strong proinflammatory activity that can dangerously affect the human body when overproduced. Since 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] IL-2 exerts both stimulatory and regulatory functions in the immune system and, together with other members of the common gamma chain (γc) cytokine family, is central to immune homeostasis. IL-2 mediates its actions by binding to the IL-2 receptor (IL-2R), which consists of a trimeric receptor composed of the IL-2Rα (CD25), IL-2Rβ (CD122), and IL-2Rγ (γc, CD132) chains or a dimeric βγ IL-2R (1, 3). Both IL-2R variants can transduce signals upon IL-2 binding. However, the trimeric αβγ IL-2R has approximately 10- to 100-fold higher affinity for IL-2 than the dimeric βγ IL-2R (3), suggesting that CD25 confers high-affinity binding of IL-2 to its receptor but is not essential for signal transduction. Trimeric IL-2R is found on activated T cells and on CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Tregs), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44 high memory phenotype (MP) CD8+, and natural killer (NK) cells are endowed with high levels of dimeric βγ IL-2R, and these cells also respond vigorously to IL-2 in vitro and in vivo.

[0041] Expression of high affinity IL-2R is important for T cells to respond to low concentrations of IL-2 that are transiently available in vivo. Expression of IL-2Rα is absent in naive and memory T cells, but is induced after antigen activation. IL-2Rβ is constitutively expressed by NK, NKT, and memory CD8+ T cells, but is also induced in naive T cells after antigen activation. γc is less tightly regulated and is constitutively expressed by all lymphoid cells. Once the high affinity IL-2R is induced by antigen, IL-2R signaling increases the expression of IL-2Rα, in part through Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism to maintain expression of the high affinity IL-2R to sustain IL-2 signaling while a source of IL-2 remains available.

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

[0043] In other words, the IL-2Rα-IL-2Rβ-IL-2 ternary complex then recruits γc through weak interactions with IL-2 and stronger interactions with IL-2Rβ, generating a stable quaternary high affinity IL-2R (Kd10-11M, i.e., 10 pM). Formation of the high affinity IL-2-IL-2R quaternary complex results in signal transduction via the tyrosine kinases Jak1 and Jak3 associated with IL-2Rβ and γc, respectively (Nelson and Willerford, 1998). The IL-2-IL-2R quaternary complex is rapidly internalized and IL-2, IL-2Rβ, and γc are rapidly degraded, whereas IL-2Rα is recycled to the cell surface (Hemar et al., 1995, Yu and Malek, 2001). Thus, their functional activity, which requires sustained IL-2R signaling, requires a continuous source of IL-2 to associate with IL-2Rα to form additional IL-2-IL-2R signaling complexes.

[0044] Interleukin-15 (IL-15), another member of the four-alpha-helical bundle family of cytokines, has also emerged as an immunomodulatory agent for cancer therapy. IL-15 is initially captured via IL-15Rα expressed on antigen-presenting dendritic cells, monocytes and macrophages. IL-15 exhibits broad activity, inducing differentiation and proliferation of T cells, B cells and natural killer (NK) cells by signaling through the IL-15 / IL-2-R-β (CD122) and common γ chain (CD132). It also mediates the differentiation and proliferation of CD8 + Enhances the cytolytic activity of T cells and sustains antigen-experienced CD8 +It induces CD44 memory T cells. IL-15 stimulates differentiation and immunoglobulin synthesis by B cells and induces maturation of dendritic cells. It does not stimulate immunosuppressive regulatory T cells (Tregs). Therefore, selective promotion of IL-15 activity in the tumor microenvironment may enhance innate and specific immunity to fight tumors (Waldmann et al., 2012). IL-15 was initially identified for its ability to stimulate IL-2-like T cell proliferation through a common receptor component (IL-2R / 15Rβ-γc) and signaling through JAK1 / JAK3 and STAT3 / STAT5. Like IL-2, IL-15 has been shown to stimulate proliferation of activated CD4-CD8-, CD4+CD8+, CD4+ and CD8+ T cells, as well as induce cytotoxic T lymphocytes and promote the development, proliferation and activation of NK cells (Waldmann et al., 1999). However, unlike IL-2, which is required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+ Treg cells and is required for the retention of these cells in the periphery, IL-15 has little effect on Tregs (Berger et al., 2009). This is important because FOXP3-expressing CD4+CD25+ Tregs inhibit effector T cells and thereby inhibit immune responses, including those directed against tumors. IL-2 also has a key role in initiating activation-induced cell death (AICD), a process that leads to the elimination of autoreactive T cells, whereas IL-15 is an antiapoptotic factor for T cells (Marks-Konczalik et al., 2000). IL-15 co-delivered with HIV peptide vaccines has been shown to promote the longevity of antigen-specific CD8+ T cells and overcome CD4+ T cell deficiencies by blocking TRAIL-mediated apoptosis (Oh et al., 2008). Furthermore, IL-15 promotes the long-term maintenance of CD8+CD44hi memory T cells (Kanegane et al., 1996).

[0045] The importance of IL-15 and IL-15Rα for the development of T cells and NK cells is - / - and IL-15 - / - This is further highlighted by the phenotype of the mice, which exhibit reduced numbers of total CD8+ T cells and are deficient in memory phenotype CD8+ T cells, NK cells, NK / T cells, and some subsets of intestinal intraepithelial lymphocytes, indicating that IL-15 provides an essential positive homeostatic function for these subsets of cells (Lodolce et al., 1996; Kennedy et al., 2000). (e.g., et al., 1998). The similarities in the phenotypes of these two strains of knockout mice suggest the importance of IL-15Rα in maintaining a physiologically relevant IL-15 signal.

[0046] IL-15 is presented in trans by the IL-15 receptor alpha chain to the IL-15Rβγc complex displayed on the surface of T cells and natural killer (NK) cells (Han et al., 2011). The IL-15Ra chain acts as a chaperone protein, stabilizing and increasing IL-15 activity (Desbois et al., 2016). Exogenous IL-15 has been shown to have limited impact in cancer patients because it relies on IL-15Ra, which is frequently reduced in cancer patients. Therefore, the fusion protein RLI, consisting of the sushi domain of IL15Ra linked to IL-15 via a linker, has been proposed as an alternative method of IL15 therapy (Bessard et al., 2009). It was found that administration of soluble IL-15 / IL-15Rα complexes greatly enhanced the serum half-life and bioavailability of IL-15 in vivo (Stoklasek et al., 2010).

[0047] In addition to its effects on T cells and NK cells, IL-15 also has several effects on other components of the immune system. IL-15 protects neutrophils from apoptosis, regulates phagocytosis, and stimulates the secretion of IL-8 and IL-1R antagonists. This works through activation of JAK2, p38 and ERK1 / 2 MAPK, Syk kinase, and NF-kB transcription factors (Pelletier et al., 2002). In mast cells, IL-15 can act as a growth factor and an apoptosis inhibitor. In these cells, IL-15 activates the JAK2 / STAT5 pathway, without the requirement for γc binding (Tagaya et al., 1996). IL-15 also induces the proliferation and differentiation of B lymphocytes and increases the secretion of immunoglobulins (Armitage et al., 1995). It also prevents Fas-mediated apoptosis and allows induction of antibody responses partially independent of CD4 help (Demerci et al., 2004; Steel et al., 2010). Monocytes, macrophages and dendritic cells efficiently transcribe and translate IL-15. They also respond to IL-15 stimulation. Macrophages respond by increasing phagocytosis, inducing expression of IL-8, IL-12 and MCP-1, and secreting IL-6, IL-8 and TNFα (Budagian et al., 2006). Dendritic cells incubated with IL-15 show maturation with increased expression of CD83, CD86, CD40 and MHC class II, are resistant to apoptosis and show enhanced secretion of interferon-γ (Anguille et al., 2009).

[0048] IL-15 has also been shown to act on non-hematopoietic cells such as muscle cells, adipocytes, endothelial cells, and neurons. IL-15 may have an anabolic effect on muscle and support the differentiation of muscle cells (Quinn et al., 1995). It can stimulate muscle cells and muscle fibers to accumulate contractile proteins and delay muscle wasting in rats with cancer-related cachexia (Figueras et al., 2004). IL-15 has also been shown to stimulate angiogenesis (Angiolillo et al., 1997) and induce the growth and survival of microglia (Hanisch et al., 1997).

[0049] Interleukin-7 (IL-7), also in the IL-2 / IL-15 family, is a well-characterized pleiotropic cytokine expressed by stromal cells, epithelial cells, endothelial cells, fibroblasts, smooth muscle cells, and keratinocytes, and after activation, by dendritic cells (Alpdogan et al., 2005). Originally described as a factor for the growth and differentiation of precursor B lymphocytes, subsequent studies have shown that IL-7 is critically involved in the development and differentiation of T lymphocytes. IL-7 signaling is essential for optimal CD8 T cell function, homeostasis, and the establishment of memory (Schluns et al., 2000), is required for the survival of most T cell subsets, and its expression has been proposed to be important for the regulation of T cell numbers.

[0050] IL-7 binds to IL-7Rα and γ cIL-7Rα binds to isodimeric receptors to form a ternary complex that plays a fundamental role in extracellular matrix remodeling, development, and homeostasis of T and B cells (Mazzucchelli and Durum, 2007). IL-7Rα also cross-reacts with thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR) to form a ternary complex to activate the TSLP pathway, leading to the proliferation of T cells and dendritic cells in humans and further development of B cells in mice (Leonard, 2002). Thus, tight regulation of the signaling cascade activated by the complex is essential for normal cell function. Understimulation of the IL-7 pathway caused by mutations in the IL-7Rα extracellular domain inhibits the development of T and B cells, and patients develop a form of severe combined immunodeficiency (SCID) (Giliani et al., 2005; Puel et al., 1998).

[0051] IL-7 has a potential role in enhancing immune reconstitution in cancer patients after cytotoxic chemotherapy. IL-7 therapy can enhance immune reconstitution and augment even limited thymic function by promoting peripheral expansion of thymic-emigrated T cells, even at low numbers. Thus, IL-7 therapy is likely to be able to restore the immune system in patients immunodepleted by cytotoxic chemotherapy (Capitini et al., 2010).

[0052] Interleukin-12 (IL-12) is a disulfide-linked heterodimer of two separately encoded subunits (p35 and p40) that are covalently linked to give rise to the so-called biologically active heterodimeric (p70) molecule (Lieschke et al., 2003). Apart from forming heterodimers (IL-12 and IL-23), the p40 subunit also exists as a monomer (p40) and as a homodimer (p40 2IL-12 is also secreted as a IL-12-binding protein (IL-12) in the host, as well as a IL-12-binding protein (IL-12-binding protein). It is known in the art that synthesis of the heterodimer as a single chain with a linker connecting the p35 and p40 subunits 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 that favor cell-mediated immunity. It has been shown that overproduction of IL-12 can be dangerous for the host, as it is involved in the pathogenesis of several autoimmune inflammatory diseases (e.g., MS, arthritis, type 1 diabetes).

[0053] The IL-12 receptor (IL-12R) is a heterodimeric complex consisting of IL-12Rβ1 and IL-12Rβ2 chains expressed on the surface of activated T cells and natural killer cells (Trinchieri et al., 2003). The IL-12Rβ1 chain 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 effects of IL-12 on cells are mainly due to activation of STAT4. IL-12 induces natural killer and T cells to produce cytokines, particularly interferon (IFN)-γ, which mediate many of the proinflammatory activities of IL-12, including differentiation of CD4+ T cells towards a Th1 phenotype ( Montepaone et al., 2014 ).

[0054] Regulatory T cells actively suppress the activation of the immune system, preventing pathological autoreactive diseases and the resulting autoimmune diseases. The development of drugs and methods to selectively activate regulatory T cells for the treatment of autoimmune diseases has been the subject of intense research and has been largely unsuccessful until the development of the present invention, which can selectively deliver active interleukins to the site of inflammation. Regulatory T cells (Tregs) are a class of CD4+CD25+ T cells that suppress the activity of other immune cells. Tregs are central to immune system homeostasis and play a major role in maintaining tolerance to self-antigens and regulating immune responses to foreign antigens. Several autoimmune and inflammatory diseases, such as type 1 diabetes (T1D), systemic lupus erythematosus (SLE), and graft-versus-host disease (GVHD), have been shown to have deficiencies in Treg cell numbers or Treg function.

[0055] As a result, there is great interest in developing therapies to increase the number and / or function of Treg cells. One method of treating autoimmune diseases that has been explored is the transplantation of autologous ex vivo expanded Treg cells (Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15). This method has shown promise in treating animal models of disease and in some early-stage human clinical trials, but requires personalized medicine with the patient's own T cells, is invasive, and is technically complex. Another method is treatment with low doses of interleukin-2 (IL-2). Treg cells characteristically express high constitutive levels of the high-affinity IL-2 receptor IL2Rαβγ, which is composed of the subunits IL2Rα (CD25), IL2Rβ (CD122), and IL2Rγ (CD132), and proliferation of Treg cells has been shown to be IL-2 dependent (Malek, TR, et al., 2010, Immunity, 33:153-65).

[0056] Conversely, immune activation has also been achieved using IL-2, and recombinant IL-2 (Proleukin®) has been approved for the treatment of certain cancers. High-dose IL-2 is used to treat patients with metastatic melanoma and metastatic renal cell carcinoma with long-term impact on overall survival.

[0057] Clinical trials of low-dose IL-2 treatment in patients with chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and HCV-associated autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg levels and signs of clinical efficacy. New clinical trials have been initiated investigating the efficacy of IL-2 in several other autoimmune and inflammatory diseases. The rationale for using so-called low-dose IL-2 was to take advantage of the high IL-2 affinity of the trimeric IL-2 receptor constitutively expressed on Tregs, while leaving other T cells that do not express the high-affinity receptor in an inactivated state. The recombinant form of IL-2 used in these trials, aldesleukin (sold as Proleukin® by Prometheus Laboratories, San Diego, CA), is associated with high toxicity. Aldesleukin, in high doses, is approved for the treatment of metastatic melanoma and metastatic renal carcinoma, but its side effects are so severe that its use is recommended only in hospitals with intensive care facilities (Web address: www.proleukin.com / assets / pdf / proleukin.pdf).

[0058] Because Treg cells respond to lower concentrations of IL-2 than many other immune cell types due to their expression of IL2Ralpha, clinical trials of IL-2 in autoimmune diseases have used lower doses of IL-2 to target Treg cells (Klatzmann D, 2015 Nat Rev Immunol. 15:283-94). However, even these low doses raised safety and tolerability issues, and the treatments used employed daily subcutaneous injections either chronically or in intermittent 5-day treatment courses. Thus, there is a need for treatments for autoimmune diseases that enhance Treg cell numbers and function, target Treg cells more specifically than IL-2, and are safer, better tolerated, and less frequently administered.

[0059] One proposed approach to improve the therapeutic index of IL-2 therapy for autoimmune diseases is to use a variant of IL-2 that is selective for Treg cells compared to other immune cells. IL-2 receptors are expressed on a wide variety of immune cell types, including T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern may contribute to its pleiotropic effects on the immune system and its high systemic toxicity. In particular, activated effector T cells express IL2Rαβγ, as do lung epithelial cells. However, activation of effector T cells is in direct opposition to the goal of suppressing and controlling the immune response, and activation of lung epithelial cells results in known dose-limiting side effects of IL-2, such as pulmonary edema. In fact, the main side effect of high-dose IL-2 immunotherapy is vascular leak syndrome (VLS), which causes intravascular fluid accumulation in organs such as the lungs and liver, leading to subsequent pulmonary edema and hepatocellular injury. There is no treatment for VLS other than discontinuing IL-2. To avoid VLS, low-dose IL-2 regimens have been tested in patients, but at the cost of suboptimal treatment outcomes.

[0060] According to the literature, VLS is thought to be caused by the release of inflammatory cytokines from IL-2-activated NK cells. However, there is some evidence that pulmonary edema results from direct binding of IL-2 to pulmonary endothelial cells expressing low to moderate levels of functional αβγ IL-2R. Moreover, pulmonary edema associated with the interaction of IL-2 with pulmonary endothelial cells was suppressed in CD25-deficient host mice by blocking binding to CD25 with an anti-CD25 monoclonal antibody (mAb) or by the use of a CD122-specific IL-2 / anti-IL-2 mAb (IL-2 / mAb) complex, thereby preventing VLS.

[0061] Treatment with interleukin cytokines other than IL-2 is more limited. IL-15 exhibits similar immune cell stimulatory activity as IL-2, but without the same inhibitory effects, making it a promising candidate for immunotherapy. Clinical trials of recombinant human IL-15 for the treatment of metastatic malignant melanoma or renal cell carcinoma showed clear changes in immune cell distribution, proliferation, and activation, suggesting potential antitumor activity (Conlon et.al.,2014). IL-15 is currently undergoing clinical trials to treat various forms of cancer. However, IL-15 treatment is known to be associated with undesirable toxic effects, such as worsening certain leukemias, graft-versus-host disease, hypotension, thrombocytopenia, and liver damage. (Mishra A.,et al.,Cance Cell,2012,22(5):645-55,Alpdogan O.et al.,Blood,2005,105(2):866-73,Conlon KC et al.,J Clin Oncol,2015,33(1):74-82).

[0062] IL-7 promotes lymphocyte development in the thymus and maintains homeostasis of naive and memory T cell survival in the periphery. It is also important for lymph node (LN) organogenesis and for the maintenance of activated T cells recruited to secondary lymphoid organs (SLOs) (Gao et al., 2013). et.al.,2015). In clinical trials of IL-7, patients receiving IL-7 showed an increase in both CD4+ and CD8+ T cells, without a significant increase in the number of regulatory T cells monitored by FoxP3 expression (Sportes et al.,2008). In clinical trials reported in 2006, 2008, and 2010, patients with various types of cancer, such as metastatic melanoma or sarcoma, were subcutaneously injected with different doses of IL-7. Little toxicity was observed, except for transient fever and mild erythema. Circulating levels of both CD4+ and CD8+ T cells were significantly increased, and Treg numbers were decreased. TCR repertoire diversity was increased after IL-7 therapy. However, the antitumor activity of IL-7 has not been fully evaluated (Gao et.al.,2015). The results suggest that IL-7 therapy may enhance and expand immune responses.

[0063] 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 stimulatory factor. By bridging innate and adaptive immunity and by potently stimulating the production of IFNγ, a cytokine that integrates natural anticancer defense mechanisms, IL-12 appeared to be an ideal candidate for tumor immunotherapy in humans. However, the severe side effects associated with systemic administration of IL-12 in clinical studies and the very narrow therapeutic index of this cytokine have significantly dampened enthusiasm for its use in cancer patients (Lasek et.al.,2014). Attempts at IL-12 therapy, where the cytokine delivery is targeted to tumors, may alleviate some of the previous problems with IL-12 therapy and are currently undergoing clinical trials for cancer.

[0064] The direct use of IL-2 as an agonist to bind IL-2R and therapeutically modulate immune responses has been problematic due to its well-documented therapeutic risks, such as its short serum half-life and high toxicity. These risks have also limited the development of therapeutics and the use of other cytokines. New forms of cytokines that reduce these risks are needed. Disclosed herein are compositions and methods comprising IL-2 and IL-15 and other cytokines, functional fragments and muteins of cytokines, and conditionally active cytokines designed to address these risks and provide needed immunomodulatory therapies.

[0065] The present invention is designed to address the shortcomings of direct IL-2 therapy and other cytokine-based therapies, for example, using cytokine blocking moieties, such as 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 beta 1, TGF beta 2, TGF beta 3), chemokines (CXC motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage colony stimulating factor (GM-CS). As used herein, "chemokines" refers to a family of small cytokines capable of inducing chemotaxis directed toward the vicinity of responsive cells. Although cytokines can provide powerful therapeutics, they are associated with undesirable effects that are difficult to manage clinically, limiting their clinical use. The present disclosure relates to new 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 above that contain cytokines or active fragments or mutant proteins 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 higher potency than the corresponding naturally occurring cytokines. As described herein, this is typically accomplished using cytokine-blocking moieties that block or inhibit the receptor activation function of the cytokine, its active fragments or mutant proteins, 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).

[0066] Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be made using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be made using recombinant DNA technology, synthetic chemistry, or a combination of these technologies, and expressed in a suitable expression system, such as CHO cells. Chimeric polypeptides can also be made by expression of a suitable nucleic acid, such as using synthetic or semi-synthetic chemical technology. 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: 193) (X is any standard amino acid) between Thr and Gly residues on a target protein, where the Thr residue transiently binds to an active site Cys residue on 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 and displaces sortase A, linking the two molecules.

[0067] To form a cytokine blocking moiety fusion protein, a cytokine polypeptide is first tagged with a polyglycine sequence at the N-terminus or, alternatively, with an LPXTG motif (SEQ ID NO: 193) at the C-terminus. The blocking moiety or other element has a peptide attached to it, respectively, which serves as an acceptor site for the tagged polypeptide. For binding to a domain carrying an LPXTG (SEQ ID NO: 193) acceptor peptide attached via its N-terminus, the polypeptide is tagged with a polyglycine stretch at its N-terminus. For binding to a domain carrying a polyglycine peptide attached via its C-terminus, the polypeptide is tagged with an LPXTG (SEQ ID NO: 193) sortase recognition sequence at its C-terminus. Upon recognition of the polyglycine and LPXTG (SEQ ID NO: 193) 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, which occurs at room temperature.

[0068] Various mechanisms can be utilized to eliminate or reduce the inhibition caused by the blocking moiety. For example, the pharmaceutical composition can include a cytokine moiety and a blocking moiety, such as a steric blocking moiety, along with a protease cleavable linker that includes a protease cleavage site located between the cytokine and the cytokine blocking moiety or within the cytokine blocking moiety. When the protease cleavage site is cleaved, the blocking moiety can dissociate from the cytokine, and the cytokine can then activate the cytokine receptor. The cytokine moiety can also be blocked by a specific blocking moiety, such as an antibody, that binds to an epitope found on the relevant cytokine.

[0069] 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 (Gly 4 Ser) n (SEQ ID NO: 195) or (Gly 3 Ser) n(SEQ ID NO: 196), where n is 1, 2, 3, 4 or 5. Typically, the sortase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 193), 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.

[0070] Thus, as detailed herein, the cytokine blocking moiety used may be a steric blocking agent. As used herein, a "steric blocking agent" refers to a polypeptide or polypeptide moiety that may be covalently linked directly or indirectly to a cytokine polypeptide, for example in the form of a chimeric polypeptide (fusion protein) or the like, via other moieties such as linkers, but is not otherwise covalently linked to the cytokine polypeptide. A steric blocking agent may be non-covalently linked 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 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 the cytokine polypeptide at a site between the steric blocking agent and the cytokine polypeptide.

[0071] 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.

[0072] In order to provide a concise disclosure of the full scope of the invention, the aspects of the invention are detailed using the cytokine IL-2 as an exemplary cytokine. However, the invention and disclosure are not limited to IL-2. It will be clear to one skilled in the art that the disclosure, including the disclosed methods, polypeptides and nucleic acids, adequately describes and enables the use of other cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFN alpha, IFN beta, IFN gamma, TNF alpha, lymphotoxin, TGF-beta 1, TGF beta 2, TGF beta 3, GM-CSF, CXCL10, CCL19, CCL20, CCL21 and functional fragments or muteins of any of the foregoing.

[0073] Various factors ensure preferential delivery and activity of IL-2 at the site of desired IL-2 activity, and severely limit systemic exposure to the interleukin through blocking and / or targeting strategies preferentially coupled with serum half-life extension strategies in which a blocked form of the interleukin circulates for an extended period of time (preferably 1-2 weeks or more) while the activated form has a typical serum half-life for an interleukin.

[0074] In comparison with its extended serum half-life form, the serum half-life of intravenously administered IL-2 is only about 10 minutes due to distribution to the extracellular space throughout the body, which is large, about 15 L in an average-sized adult. IL-2 is then metabolized by the kidney, with a half-life of about 2.5 hours. (Smith, K. "Interleukin 2 immunotherapy". Therapeutic Immunology 240 (2001)). In other measurements, IL-2 has a very short plasma half-life of 85 minutes when administered intravenously and 3.3 hours when administered subcutaneously (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). In some embodiments of the invention, the half-life extension element is linked to the interleukin via a linker and cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to 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 and a half-life of hours rather than weeks. Furthermore, exposure to the active cytokine is limited to the site of the desired cytokine activity (e.g., the site of inflammation or tumor), reducing systemic exposure to the active cytokine and associated toxicity and side effects.

[0075] Other cytokines contemplated by the present invention have similar pharmacology to IL-2 (e.g., IL-15 reported in Blood 2011 117:4787-4795; doi:doi.org / 10.1182 / blood-2010-10-311456), and thus the design of the present invention addresses the shortcomings of using these agents directly, providing chimeric polypeptides that have longer half-lives and / or can be directed to the desired site of activity (e.g., sites of inflammation or tumors).

[0076] If desired, IL-2 can be engineered to bind generally to the IL-2R complex or to specifically bind to one of the three IL-2R subunits with an affinity that differs from that of the corresponding wild-type IL-2, e.g., to selectively activate Tregs or Teffs. For example, an IL-2 polypeptide that is said to have a higher affinity for the trimeric form of the IL-2 receptor compared to the dimeric beta / gamma form of the IL-2 receptor compared to wild-type IL-2 can be obtained by modifying SEQ ID NO: 1 (the mature IL-2 protein comprising amino acids 21-153 of human IL-2 having Uniprot accession number P60568-1) with a set of mutations, namely: (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, Q74P, and I128T; 3V, and K76E, (f) V69A, Q74P, T101A, and T133N, (g) N30S, V69A, Q74P, and I128A, (h) V69A, Q74P, N88D, and S99P, (i) N30S, V69A, Q74P, and I128T, (j) K9T, Q11R, K35R, V6 9A, and Q74P, (k) A1T, M46L, K49R, E61D, V69A, and H79R, (l) K48E, E68D, N71T, N90H, F103S, and I114V, (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A, (n) E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T, (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H, (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T, (q) N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T, (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T, and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V.This method can also be applied to prepare muteins of other cytokines, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs, such as IFN alpha, IFN beta, and IFN gamma), tumor necrosis factors (e.g., TNF alpha, lymphotoxin), transforming growth factors (e.g., TGF beta 1, TGF beta 2, TGF beta 3), and granulocyte macrophage colony stimulating factor (GM-CS). For example, muteins with desired binding affinity for their cognate receptors can be prepared.

[0077] As noted above, any of the mutant IL-2 polypeptides disclosed herein can include, or be limited to, the sequences set forth or can otherwise be identical to SEQ ID NO: 1. Additionally, any of the mutant IL-2 polypeptides disclosed herein can optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine) and / or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO:1.

[0078] Another way to improve the therapeutic index of IL-2 therapy is to optimize the pharmacokinetics of the molecule to maximize activation of Treg cells. Early studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro required a minimum of 5-6 hours of exposure to effective concentrations of IL-2 (Cantrell, DA, et.al., 1984, Science, 224:1312-1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes intravenously and 3.3 hours subcutaneously (Kirchner, GI, et al., 1998, Br J Clin Pharmacol. 46:5-10). Due to its short half-life, maintaining circulating IL-2 at levels above those required to stimulate T cell proliferation for the time required requires high doses that result in peak IL-2 levels well above the EC50 of Treg cells or requires frequent dosing. These high IL-2 peak levels can activate the IL2Rβγ receptor and can result in other unintended or deleterious effects, such as VLS, as described above. IL-2 analogs, or multifunctional proteins with longer circulating half-lives than IL-2, in which IL-2 is linked to a domain that allows binding to the FcRn receptor, can achieve a desired drug concentration at lower doses and lower peak levels than IL-2 for a specified period of time. Thus, such IL-2 analogs require lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of IL-2 drugs is also more easily tolerated by patients. Therapies with these characteristics clinically translate into improved pharmacological efficacy of the therapy, reduced toxicity, and improved patient compliance. Alternatively, IL-2 or IL-2 muteins (herein referred to as "IL-2 * ") can be selectively directed to the intended site of action (e.g., a site of inflammation or a tumor). This targeting can be achieved by one of several strategies, such as the addition of a domain to the administered drug that contains a blocker of IL-2 (or a mutant protein) that is shed, or by targeting the domain, or by a combination of the two.

[0079] In some embodiments, IL-2 * Partial agonists can be tailored to bind with higher or lower affinity depending on the desired target, e.g., IL-2 * can be engineered to bind with enhanced affinity to one of the receptor subunits and not to the other subunits. These types of partial agonists, unlike full agonists or full antagonists, offer the ability to tune the signaling properties to an amplitude that elicits desired functional properties without meeting a threshold of undesirable properties. Given the specific activity of partial agonists, the repertoire of IL-2 variants can be engineered to exhibit even finer degrees of characteristic signaling activity, ranging from near-full to partial agonism to full antagonism.

[0080] In some embodiments, IL-2 * In some embodiments, the IL-2 * has a higher affinity for IL-2Rα than wild-type IL-2. * has an altered affinity for IL-2Rβ. In one embodiment, IL-2 * In another embodiment, IL-2Rα has enhanced binding affinity to IL-2Rβ, e.g., the N-terminus of IL-2Rβ, thereby eliminating the functional requirement for IL-2Rα. * was engineered to have increased binding affinity for IL-2Rβ but reduced binding to IL-2Rγ, and is therefore defective in IL-2Rβγ heterodimerization and signaling.

[0081] Blocking moieties, detailed below, can also be used to promote binding to or activation of one or more receptors. In one embodiment, a blocking moiety is added such that IL-2Rβγ binding or activation is blocked but IL-2Rα binding or activation is not altered. In another embodiment, a blocking moiety is added such that IL-2Rα binding or activation is reduced. In another embodiment, a blocking moiety is added such that binding and / or activation of all three receptors is inhibited. This blockage can be relieved by removal of the blocking moiety in certain circumstances, for example, by proteolytic cleavage of a linker linking one or more blocking moieties to the cytokine.

[0082] Similar methods can be applied to improve other cytokines, particularly for use as immune stimulants, such as for cancer therapy. For example, in this embodiment, cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, 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-49, IL-4 The pharmacokinetics and / or pharmacodynamics of 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 maximally activate effector cells (e.g., effector T cells, NK cells) and / or cytotoxic immune response promoting cells at the desired site of activity, such as within a tumor, preferably not systemically, (e.g., induce maturation of dendritic cells).

[0083] Thus, provided herein are pharmaceutical compositions comprising at least one cytokine polypeptide, such as 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 beta 1, TGF beta 2, TGF beta 3), chemokines (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte macrophage colony stimulating factor (GM-CS), or functional fragments or muteins of any of the foregoing. The polypeptides typically also include at least one linker amino acid sequence, which, in certain embodiments, is susceptible to cleavage by endogenous proteases. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 197), IPVSLRSG (SEQ ID NO: 198), VPLSLYSG (SEQ ID NO: 199), or SGESPAYYTA (SEQ ID NO: 200). In other embodiments, the chimeric polypeptide further comprises a blocking moiety capable of blocking the activity of the interleukin polypeptide, such as a steric blocking polypeptide moiety. 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 the cytokine receptor, and in certain embodiments, the blocking moiety comprises a protease-cleavable domain. In some embodiments, blocking and reducing cytokine activity is easily achieved by attaching additional domains to the N-terminus 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 excised or released, it becomes impossible to achieve blockade of cytokine activity.

[0084] A pharmaceutical composition, for example, a chimeric polypeptide, can comprise two or more cytokines, which can be the same cytokine polypeptide or different cytokine polypeptides. For example, two or more different types of cytokines have complementary functions. In some examples, the first cytokine is IL-2 and the second cytokine is IL-12. In some embodiments, each of two or more different types of cytokine polypeptides has the activity of regulating the activity of other cytokine polypeptides. In some examples of a chimeric polypeptide containing two cytokine polypeptides, the first cytokine polypeptide is T cell activation and the second cytokine polypeptide is non-T cell activation. In some examples of a chimeric polypeptide containing two cytokine polypeptides, the first cytokine is a chemoattractant, for example, CXCL10, and the second cytokine is an immune cell activator.

[0085] Preferably, the cytokine polypeptides (including functional fragments) contained in the fusion proteins disclosed herein are not mutated or engineered to modify the properties of naturally occurring cytokines such as receptor binding affinity and specificity or serum half-life. However, changes in the amino acid sequence from a naturally occurring (including wild-type) cytokine are permitted, for example, to facilitate cloning and to achieve the desired expression level.

[0086] Blocking moiety A blocking moiety can be any moiety that inhibits the ability of a cytokine to bind and / or activate its receptor. A blocking moiety can inhibit the ability of a cytokine to bind and / or activate its receptor by sterically blocking 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 that bind cytokines, single-chain variable fragments (scFv), single domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (VL) and variable domains of camelid nanobodies (VHH)), dAbs, etc., can also be used. 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 SpA, GroEL, fibronectin, lipocalin, and CTLA4 scaffolds. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of a cytokine to its cognate receptor. 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 a cytokine to its receptor. 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 the heavy chain variable domain (VH), light chain variable domain (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 SpA, GroEL, fibronectin, lipocalin and CTLA4 scaffolds.

[0087] In illustrative examples, where IL-2 is the cytokine in the chimeric polypeptide, the blocking moiety can be a full-length or fragment or mutein of the IL-2 receptor alpha chain (IL-2Rα) or IL-2 receptor beta (IL-2Rβ) or gamma chain (IL-2Rγ), an anti-IL-2 single domain antibody (dAb) or scFv, a Fab, an anti-CD25 antibody or fragment thereof, and an anti-HAS dAb or scFv, etc.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 4. The fusion protein of any one of aspects 1-2, wherein the binding moiety is capable of binding to a bulk serum protein.

[0092] 5. The fusion protein of any one of aspects 1-3, 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.

[0093] 6. The fusion protein of any one of aspects 1 to 4, wherein the binding moiety further comprises a complementarity determining region (CDR).

[0094] 7. The fusion protein of aspect 5, wherein the binding moiety is capable of binding to the bulk serum protein.

[0095] 8. The fusion protein of aspect 6, wherein said bulk serum protein is a half-life extending protein.

[0096] 9. The fusion protein of aspect 6 or 7, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0097] 10. The fusion protein according to 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.

[0098] 11. The fusion protein according to any one of aspects 1 to 9, wherein the cleavable linker comprises a cleavage site.

[0099] 12. The fusion protein according to aspect 10, wherein the cleavage site is recognized by a protease.

[0100] 13. The fusion protein of aspect 11, wherein the binding moiety is conjugated to the cytokine.

[0101] 14. The fusion protein of aspect 11 or 11, wherein the binding moiety is covalently linked to the cytokine.

[0102] 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.

[0103] 16. The fusion protein of any one of aspects 11-14, wherein the non-CDR loop provides a specific binding site for binding of the moiety to the cytokine.

[0104] 17. The fusion protein according to 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.

[0105] 18. The fusion protein according to any one of aspects 1 to 16, wherein the cytokine binds to a cytokine receptor.

[0106] 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.

[0107] 20. The fusion protein according to any one of aspects 1 to 18, wherein the cleavable linker comprises a cleavage site.

[0108] 21. The fusion protein according to aspect 20, wherein the cleavage site is recognized by a protease.

[0109] 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 aspartic peptide lyase.

[0110] 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 amyloid-like protease. 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 according to embodiment 21, which is recognized by MP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1 beta 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).

[0111] 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.

[0112] 25. The conditionally active binding protein according to aspect 24, wherein the binding moiety is capable of binding to a half-life extending protein.

[0113] 26. The conditionally active binding protein according to aspect 24 or 25, wherein said binding moiety is a natural peptide, a synthetic peptide, an engineered scaffold, or an engineered serum bulk protein.

[0114] 27. The conditionally active binding protein according to aspect 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.

[0115] 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.

[0116] 29. The conditionally active binding protein according to any one of aspects 24 to 28, wherein said binding moiety further comprises a complementarity determining region (CDR).

[0117] 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.

[0118] 31. The conditionally active binding protein of aspect 30, wherein the bulk serum protein is albumin, transferrin, factor XIII, or fibrinogen.

[0119] 32. The conditionally active binding protein according to any one of aspects 29-31, wherein said CDRs provide specific binding sites for said bulk serum protein or said immunoglobulin light chain, or any combination thereof.

[0120] 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.

[0121] 34. The conditionally active binding protein according to any one of aspects 29-33, wherein said non-CDR loops provide specific binding sites for binding of said binding moiety to said cytokine.

[0122] 35. The conditionally active binding protein of any one of aspects 24-34, wherein the cytokine binds to a cytokine receptor.

[0123] 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.

[0124] 37. The conditionally active binding protein according to any one of aspects 24 to 36, wherein the cleavable linker comprises a cleavage site.

[0125] 38. The conditionally active binding protein according to embodiment 37, wherein the cleavage site is recognized by a protease.

[0126] 39. The conditionally active binding protein according to embodiment 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 aspartic peptide lyase.

[0127] 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, actinomycin-like protease, and cytochrome P450. Nidane, 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 (PSA, hK3), interleukin-1β-converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptida 39. The conditionally active binding protein according to embodiment 38, which 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).

[0128] 41. The conditionally active binding protein according to aspect 24, further comprising a half-life prolonging domain linked to said binding moiety, wherein said half-life prolonging domain provides a safety switch for said binding protein, and 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.

[0129] 42. The conditionally active binding protein according to embodiment 41, wherein said cleavage of the linker is in the tumor microenvironment.

[0130] 43. A conditionally active binding protein comprising a binding moiety that binds to a cytokine via a non-CDR loop within the binding moiety, said binding moiety further linked to a half-life prolonging domain and comprising a cleavable linker, said binding protein having an extended half-life prior to its activation by cleavage of said linker, and upon activation, said binding moiety and said half-life prolonging domain are separated from said cytokine, and said binding protein in its activated state does not have an extended half-life.

[0131] 44. The conditionally active binding protein according to aspect 43, wherein said cleavage of the linker is in the tumor microenvironment.

[0132] In vivo half-life extension element Preferably, the chimeric polypeptide comprises an in vivo half-life extension 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 extension element" is 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 the parameters of absorption, biodistribution, metabolism, and elimination. An exemplary method of improving the pK of a polypeptide is by expression of an element of the polypeptide chain that binds to a receptor that is recycled to the plasma membrane of the cell rather than being degraded in lysosomes, such as the FcRn receptor and the 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, which is a function of their ability to bind to a receptor that is 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:2) can also be directly or via a short linker to the pharmaceutical composition. Fragments of HSA can also be used. HSA and its fragments can function both as blocking moieties and as half-life extension elements. Human IgG and Fc fragments can also perform similar functions.

[0133] The serum half-life extending element can also be an antigen-binding polypeptide that binds to a protein with a long serum half-life, such as serum albumin, transferrin, etc. Examples of such polypeptides include antibodies and fragments thereof, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies (such as the heavy chain variable domain (VH), the light chain variable domain (VL) and the 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 a desired receptor, ligand-binding portions of a receptor, lectins, and peptides that bind to or associate with one or more target antigens.

[0134] Some preferred serum half-life extenders are polypeptides that include a complementarity determining region (CDR) and, optionally, a non-CDR loop. Advantageously, such serum half-life extenders can extend the serum half-life of a cytokine and also function as a cytokine inhibitor (e.g., via steric blocking, non-covalent interactions, or a combination thereof) and / or as a targeting domain. In some cases, the serum half-life extender is a domain derived from an immunoglobulin molecule (Ig molecule) or is an engineered protein scaffold that mimics the structure and / or binding activity of an antibody. The Ig can be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chain of an Ig molecule folds into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute the "complementarity determining region" (CDR) 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 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 moiety 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 (CDR1, CDR2, CDR3) of the V domains form a cluster at one end of a beta barrel. The CDRs are the loops connecting the beta strands BC, C'-C", and FG of the immunoglobulin fold, while the lower loops connecting the 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 disclosure, at least some amino acid residues of the constant domains, CH1, CH2, or CH3, are part of the "non-CDR loops" of the binding moieties described herein. The non-CDR loops, in some embodiments, include one or more of the following: 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.

[0135] Within the variable domain, CDRs are believed to be involved in antigen recognition and binding, and FR residues are considered as the scaffold of CDRs. However, in certain cases, some FR residues play an important role 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 antigens, and therefore they are part of the binding site, and some of these residues are adjacent to CDRs. Other residues are adjacent to CDRs, but are adjacent in the 3-D structure of the molecule, such as loops of heavy chains. Serum half-life extending domains (e.g., domains that contain CDRs) can include 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.

[0136] 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 antigen peptide is inserted into the non-CDR loop. In some examples, the non-CDR loop is replaced with an antigen peptide. In some cases, the modification to generate an antigen binding site is only in 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, namely, 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.

[0137] 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, for example, via steric occlusion, via specific intermolecular interactions, or a combination of both.

[0138] In some embodiments, the serum half-life extending element non-covalently binds directly to the cytokine and inhibits its activity.

[0139] In certain examples, the 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, the 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, the binding moiety binds to a bulk serum protein, such as albumin, through its AB, CC', C"D, and EF loops and binds to a cytokine through its BC, C'C", and FG loops. In certain examples, the 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 binds to a cytokine through one or more of the BC, C'C", and FG loops.

[0140] The binding moiety is any kind 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.

[0141] In some cases, the serum half-life extension element is linked to the cytokine domain via its non-CDR loop, and the cytokine domain is further connected to the targeting domain described herein. In some cases, the serum half-life extension element comprises a binding site for a bulk serum protein. In some embodiments, the CDR provides a binding site for a 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 a binding site for an immunoglobulin light chain, such as an Igκ free light chain or an Igλ free light chain.

[0142] 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-2. In this example, the binding site for serum albumin can be formed by the CDRs of the serum albumin binding domain.

[0143] The serum half-life extending element can be any kind of binding domain, including but not limited to domains from monoclonal, polyclonal, recombinant, human, 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 nanobody (VHH). In other embodiments, the binding moiety is a non-Ig binding domain, i.e., anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, and antibody mimetics such as monobodies.

[0144] In other embodiments, the serum half-life extending element can 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 peptide that is predominantly, i.e., greater than 50%, -OCH 2 CH 2 -means a polymer containing repeating subunits. With respect to specific forms, PEG can be of 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 can be linear (e.g., capped ends, e.g., alkoxy PEG or bifunctional PEG), branched or multi-armed (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star) structures, each with or without one or more degradable bonds. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns and can be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating 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 that includes amino acids having side chains containing amine, sulfhydryl, carboxylic acid, or hydroxyl functional groups, such as cysteine, lysine, asparagine, glutamine, theonine, tyrosine, serine, aspartic acid, and glutamic acid.

[0145] Targeting and Retention Domains In certain applications, it may be desirable to maximize the time that the construct is present at its desired site in the body. This can be achieved by including an additional domain in the chimeric polypeptide (fusion protein) to affect its movement in the body. For example, the chimeric nucleic acid can encode a domain that directs the polypeptide to a site in the body, such as a tumor cell or site of inflammation (this domain is referred to as a "targeting domain") and / or a domain that retains the polypeptide at a site in the body, such as a tumor cell or site of inflammation (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 are specific for a protease-rich environment. In some embodiments, the encoded targeting domain and / or retention domain are specific for regulatory T cells (Tregs), for example targeting the CCR4 receptor or the CD39 receptor. Other suitable targeting and / or retention domains include those with cognate ligands overexpressed in inflamed tissues, such as the IL-1 receptor, or the IL-6 receptor. In other embodiments, suitable targeting and / or retention domains include those with cognate ligands overexpressed in tumor tissues, such as the Epcam, CEA, or mesothelin. In some embodiments, the targeting domain is linked to the interleukin via a linker and is cleaved (e.g., by an inflammation- or cancer-specific protease) at the site of action, 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 (e.g., by an inflammation- or cancer-specific protease) at the site of action, allowing the cytokine to remain at the desired site.

[0146] 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, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins that include 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.

[0147] Suitable targeting and / or retention domains include antigen-binding domains, such as antibodies and fragments thereof, such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single chain variable fragments (scFv), single domain antibodies (such as the heavy chain variable domain (VH), light chain variable domain (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 binding domains based on anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, Kunitz domain peptides, monobodies, and 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.

[0148] In some embodiments, the targeting domain and / or retention domain specifically binds to a cell surface molecule. In some embodiments, the targeting domain and / or retention domain specifically binds to a tumor antigen. In some embodiments, the targeting polypeptide specifically and independently binds to a tumor antigen selected from at least one of fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signaling agent 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the targeting polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1.

[0149] 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, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, PSMA, CD38, BCMA, and CEA, 5T4, AFP, B7-H3, cadherin-6, CAIX, CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33, CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, These include DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16E6, HPV-16E7, 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.

[0150] 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.

[0151] The target and / or retention antigen may be a cell surface molecule, such as a protein, lipid, or polysaccharide. In some embodiments, the target and / or retention antigen is 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 may 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.

[0152] 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.

[0153] Linker As mentioned above, the pharmaceutical composition includes one or more linker sequences. The linker sequence serves to provide flexibility between polypeptides, for example, to allow the blocking moiety to inhibit the activity of the cytokine polypeptide. The linker sequence can be located between any or all of the cytokine polypeptide, the serum half-life extension element, and / or the blocking moiety. As described herein, at least one of the linkers is protease cleavable and contains a cleavage site(s) for the desired protease(s). Preferably, the desired protease is enriched or selectively expressed in the desired site of cytokine activity (e.g., tumor microenvironment). Thus, the fusion protein is preferentially or selectively cleaved at the site of the desired cytokine activity.

[0154] Suitable linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0155] It will be appreciated that the orientation of the components of the pharmaceutical composition is largely a matter of design choice, with multiple orientations possible and all intended to be encompassed by the present disclosure, for example, a blocking moiety can be located at the C-terminus or N-terminus of the cytokine polypeptide.

[0156] Proteases known to be associated with diseased 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, Synth-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 metalloprotease (MMP), MMP1, MMP2 , MMP3, MMP8, MMP9, MMP13, MMP11, MMP14, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific antigen (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-a), dipeptidyl peptidase, meprin, granzyme and dipeptidyl peptidase IV (DPPIV / CD26). Proteases capable of cleaving the amino acid sequence encoded by the chimeric nucleic acid sequence provided herein may be selected from the group consisting of, for example, prostate-specific antigen (PSA), matrix metalloproteases (MMPs), A distigrin and metalloproteases (ADAMs), plasminogen activators, cathepsins, caspases, tumor cell surface proteases, and elastases. The MMP can be, for example, matrix metalloprotease 2 (MMP2) or matrix metalloprotease 9 (MMP9).

[0157] Proteases useful in the methods disclosed herein are set forth in Table 1, with exemplary proteases and their cleavage sites set forth in Table 1a. Table 1. Proteases related to inflammation and cancer [Table 1-1] [Table 1-2] [Table 1-3] Table 1a: Exemplary proteases and protease recognition sequences [Table 1a-1] [Table 1a-2]

[0158] Provided herein is a pharmaceutical composition comprising the polypeptide sequence.As with all peptides, polypeptides, and proteins (including their fragments), it is understood that additional modifications may occur in the amino acid sequence of chimeric polypeptides (amino acid sequence variants) that do not alter the properties or functions of the peptide, polypeptide, or protein.Such modifications include conservative amino acid substitutions, which are discussed in more detail below.

[0159] The compositions provided herein have a desired function. The compositions are comprised of at least a cytokine polypeptide, such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IFNa, or IFNg, or a chemokine, such as CXCL10, CCL19, CCL20, or CCL21, a blocking moiety, such as 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, such as an IL-2 mutein, is provided as an active agent. A blocking moiety is provided to block the activity of an interleukin. A linker polypeptide, such as 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 an interleukin polypeptide. In some embodiments, a 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 or tumor-specific protease) to liberate full cytokine activity at the desired site.

[0160] The protease cleavage site may be a naturally occurring protease cleavage site or an artificially engineered protease cleavage site. The artificially engineered protease cleavage site may be cleaved by two or more proteases specific to the desired environment in which cleavage occurs, such as a tumor. The protease cleavage site may be cleavable by at least one protease, at least two proteases, at least three proteases, or at least four proteases.

[0161] In some embodiments, the linker is a glycine-glycine, a sortase recognition motif, or a sortase recognition motif and a peptide sequence (Gly 4 Ser) n (SEQ ID NO: 195) or (Gly 3 Ser)n (SEQ ID NO: 196), where n is 1, 2, 3, 4, or 5. In one embodiment, the sortase recognition motif comprises the peptide sequence LPXTG (SEQ ID NO: 193), 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.

[0162] Cleavage and Inducibility As described herein, the activity of the cytokine polypeptide is attenuated in the fusion protein, and upon cleavage by a protease at the desired site of activity, such as within the tumor microenvironment, a form of cytokine is released from the fusion protein that is much 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 times, at least about 50 times, at least about 100 times, at least about 250 times, at least about 500 times, or at least about 1000 times 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 when the molecular entity contains substantially identical amino acids to the cytokine polypeptide, is substantially free of additional amino acids, and is not associated (covalently or non-covalently) with other molecules. Optionally, the cytokine polypeptide as a separate molecular entity may include some additional amino acid sequence, such as a tag or a short sequence to aid in expression and / or purification.

[0163] 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.

[0164] 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 have a different amino acid sequence from the naturally occurring protein, so long as the desired function is maintained. It is understood that one way 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, 99 percent identity to the chimeric polypeptides provided herein. For example, provide a polypeptide or nucleic acid that has at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity with any of the sequences of nucleic acids or polypeptides described herein.Those skilled in the art will easily understand how to determine the identity of two polypeptides or two nucleic acids.For example, identity can be calculated after aligning two sequences so that identity is at its highest level.

[0165] Different methods for calculating identity can be performed by publicly available algorithms. Optimal alignment of sequences for comparison is performed by the method of Smith and Waterman Adv. Appl. Math. 2:482 (1981), by the local identity algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search methods of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by 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 test validation.

[0166] 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. Although 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 is disclosed herein.

[0167] Modifications of proteins include modifications of amino acid sequences. Modifications in amino acid sequences can occur naturally as allelic variations (e.g., by genetic polymorphisms), can occur due to environmental influences (e.g., by 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. Modifications of amino acid sequences typically fall into one or more of three types: substitution, insertion, or deletion modifications. Insertions include fusions at the amino and / or carboxyl termini, as well as intrasequence insertions of single or multiple amino acid residues. Insertions are usually smaller than insertions of amino or carboxyl termini fusions, e.g., insertions of about 1-4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about 2-6 residues are deleted at any one site in the protein molecule. Amino acid substitutions are typically of a single residue, but can occur at several different positions at once, with insertions usually on about 1-10 amino acid residues and deletions ranging from about 1-30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletion of two residues or insertion of two residues. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final construct. The mutations must not place the sequence out of reading frame and preferably do not give rise to complementary regions that may generate secondary mRNA structures. Substitutional modifications are those in which at least one residue has been removed and a different residue has been inserted in its place. Such substitutions are generally made according to Table 2 below and are called conservative substitutions. Table 2. Exemplary Amino Acid Substitutions [Table 2-1] [Table 2-2]

[0168] Modifications, including specific amino acid substitutions, are made by known methods. For example, modifications are made by site-specific mutagenesis of nucleotides in DNA encoding the polypeptide, thereby generating DNA encoding the modifications, followed by expressing 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.

[0169] 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 in the complementarity determining regions (CDRs). Such random mutations can be introduced using a variety of techniques, such as radiation, chemical mutagens, error-prone PCR, etc. Multiple rounds of mutation and selection can be performed, for example, using phage display, etc.

[0170] 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 the therapeutic uses of such DNA and RNA molecules.

[0171] 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.

[0172] In some embodiments, the fusion protein comprises a cytokine, a blocking moiety, and a half-life extending element. In some embodiments, the cytokine is located between the half-life extending element and the blocking moiety. In some embodiments, the cytokine is N-terminal to the blocking moiety and the half-life extending element. In some such embodiments, the cytokine is proximal to the blocking moiety, and in some such embodiments, the cytokine is proximal to the half-life extending element. At least one protease cleavable linker must be included in all embodiments so that the cytokine may be active upon cleavage. In some embodiments, the cytokine 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.

[0173] In some embodiments, the blocking domain used is capable of extending half-life and the cytokine is positioned between two such blocking domains, hi some embodiments, the cytokine is positioned between two blocking domains, one of which is capable of extending half-life.

[0174] In some embodiments, the two cytokines are included in the same construct. In some embodiments, each cytokine has two blocking domains connected (total of three in one molecule), and there is one blocking domain between the two cytokine domains. In some embodiments, one or more additional half-life extension domains may be included to optimize pharmacokinetic properties. In some cases, it is beneficial to include two of the same cytokine to promote dimerization. One example of a cytokine that functions as a dimer is IFNγ.

[0175] In some embodiments, the three cytokines are contained in the same construct, hi some embodiments, the third cytokine may function to block the other two cytokines in place of a blocking domain between the two cytokines.

[0176] Preferred half-life extending elements for use in the fusion protein are human serum albumin (HSA), an antibody or antibody fragment that binds serum albumin (e.g., scFV, dAb), a 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 activation or activation of the cytokine receptor binding, another cytokine, or a fragment of any of the foregoing. In preferred embodiments that include 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.

[0177] Therapeutic Methods and Pharmaceutical Compositions Further provided are methods for treating a subject having or at risk of developing a disease or disorder, such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or a 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 cytokine, a fragment or a mutant protein thereof, which is activated at a site of inflammation or a tumor. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or a mutant protein thereof, and a serum half-life extending element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or a mutant protein thereof, and a blocking moiety, such as a steric blocking polypeptide, which can sterically block the activity of the cytokine polypeptide, a fragment or a mutant protein thereof. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or a mutant protein thereof, a blocking moiety, and a serum half-life extending element.

[0178] Inflammation is part of the complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, and irritants, and is a defense reaction involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of 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, allergy, 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.

[0179] The pharmaceutical composition may include one or more protease cleavable linker sequences. The linker sequence serves to provide flexibility between the polypeptides so that each polypeptide can inhibit the activity of the first polypeptide. The linker sequence may be located between any or all of the cytokine polypeptide, fragment or mutein thereof, the blocking moiety, and the serum half-life extension element. Optionally, the composition includes two, three, four, or five linker sequences. The linker sequence, the two, three, or four linker sequences may be the same or different linker sequences. In one embodiment, the linker sequence is selected from the group consisting of GGGGS (SEQ ID NO: 201), GSGSGS (SEQ ID NO: 202), or G(SGGG). 2 SGGT (SEQ ID NO: 203). In another embodiment, the linker comprises a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 197), IPVSLRSG (SEQ ID NO: 198), VPLSLYSG (SEQ ID NO: 199, and SGESPAYYTA (SEQ ID NO: 200).

[0180] 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 metalloprotease (MMP), plasminogen activator, cathepsin, caspase, tryptase, or a tumor cell surface protease.

[0181] Suitable linkers can be of different lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, 2 amino acids to 15 amino acids, 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids.

[0182] Further provided is a method of treating a subject having or at risk of developing cancer. The method comprises administering to a subject in need thereof an effective amount of the chimeric polypeptide (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 cancer. The pharmaceutical composition preferably comprises a blocked cytokine, fragment or mutant protein thereof, which is activated at the tumor site. Preferably, the tumor is a solid tumor. The cancer can be, but is not limited to, colon cancer, lung cancer, melanoma, sarcoma, renal cell carcinoma, and breast cancer.

[0183] The method may further include administering one or more additional agents to treat the cancer, such as, for example, 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, and the like.

[0184] Provided herein is a pharmaceutical formulation or composition containing a chimeric polypeptide and a pharmacologically acceptable carrier. The compositions provided herein are suitable for administration in vitro or in vivo. A pharmacologically acceptable carrier refers to a material that is not biologically or otherwise undesirable, i.e., such a material is administered to a subject without causing undesirable 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 deleterious side effects in the subject.

[0185] 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 make the formulation isotonic, but the formulation can be hypertonic or hypotonic, if desired. 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, for example, films, liposomes, or microparticles. Certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition to be administered. The carrier is one suitable for administration of the chimeric polypeptide or a nucleic acid sequence encoding the chimeric polypeptide to a human or other subject.

[0186] The pharmaceutical formulation or composition is administered in a number of ways, depending on whether local or systemic treatment is desired and on the area to be treated. The composition is administered via any of a number of 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 composition is administered locally (non-systemically), such as intratumorally, intraarticularly, intrathecally, etc.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] Optionally, the chimeric polypeptide or the nucleic acid sequence encoding the chimeric polypeptide is administered by a vector. There are several compositions and methods that can be used to deliver nucleic acid molecules and / or polypeptides to cells in vitro or in vivo, for example, via expression vectors. These methods and compositions can be broadly divided into two types: viral delivery systems and non-viral delivery systems. Such methods are well known in the art and can be easily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express and preferably secrete the encoded chimeric polypeptide, or to deliver nucleic acids therapeutically to a subject. The components of the chimeric nucleic acids disclosed herein are typically operably linked in frame to encode a fusion protein.

[0191] As used herein, a plasmid or viral vector is an entity that transports the disclosed nucleic acid to a cell in an undegraded state and contains a promoter that results in expression of the nucleic acid molecule and / or polypeptide in the cell to which it is delivered. Viral vectors include, for example, adenoviruses, adeno-associated viruses, herpes viruses, vaccinia viruses, polio viruses, Sindbis, and other RNA viruses, including those that have 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 vectors and methods for their production. The construction of replication-defective 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 airway epithelium, 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.

[0192] 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 making and using virus-like particles are described, for example, in Garcea and Gissmann, Current Opinion in Biotechnology 15:513-7 (2004).

[0193] 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).

[0194] The provided polypeptides can be delivered by exoskeletal aggregates. Methods for making and using exoskeletal aggregates are described in International Publication No. WO2006 / 110728.

[0195] Non-viral delivery methods can include expression vectors that contain a nucleic acid sequence encoding a nucleic acid molecule and a 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, for example, plasmids, artificial chromosomes, BACs, YACs, or PACs. Numerous vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Pal Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to generate chimeric polypeptides by expression in suitable host cells, such as CHO cells.

[0196] Preferred promoters controlling transcription from vectors in mammalian host cells may be derived from a variety of sources, for example from the genomes of viruses such as polyomavirus, 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.

[0197] 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 within introns or within the coding sequence itself. They are usually 10-300 base pairs (bp) in length and function in cis. Enhancers usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. Many enhancer sequences are known from mammalian genes (globin, elastase, albumin, fetoprotein, and insulin), and enhancers from eukaryotic cell viruses are typically used for general expression. Preferred examples are 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.

[0198] The promoter and / or enhancer may be inducible (e.g., chemically or physically regulated). Chemically regulated promoters and / or enhancers may be regulated, for example, by the presence of alcohol, tetracycline, steroids, or metals. Physically regulated promoters and / or enhancers may be regulated, for example, by environmental factors such as temperature and light. Optionally, the promoter and / or enhancer region may act as a constitutive promoter and / or enhancer to maximize expression of the region of the transcription unit to be transcribed. In 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).

[0199] A vector can also include, for example, an origin of replication and / or a marker. A marker gene can confer a selectable phenotype, such as antibiotic resistance, to a cell. The marker product is used to determine whether the vector has been delivered to a cell and, once delivered, whether it is 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 to a mammalian host cell, the transformed mammalian host cell can survive when placed under selection pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence 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 a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including either the carboxyl or amino terminus.

[0200] 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 suggest a particular size or number of amino acids that make up the molecule, and that the peptides of the present invention can contain up to a few or more amino acid residues. As used throughout, the subject can be a vertebrate, and 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 imply a particular age or sex. Thus, adult and newborn subjects of either sex are intended to be encompassed. 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 terms patient or subject include human subjects and veterinary subjects.

[0201] 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.

[0202] The methods and medicaments described herein are useful for both preventive and therapeutic treatments. For preventive use, a therapeutically effective amount of the chimeric polypeptides or chimeric nucleic acid sequences encoding the chimeric polypeptides described herein is administered to a subject before onset (e.g., before obvious signs of cancer or inflammation) or during early onset (e.g., at early signs and symptoms of cancer or inflammation). Prophylactic administration can be performed from days to 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 the chimeric polypeptides or nucleic acid sequences encoding the chimeric polypeptides described herein to a subject after the diagnosis or onset of cancer or inflammation (e.g., autoimmune disease). Prophylactic use can also be applied when a patient is undergoing a treatment in which inflammation is expected, such as chemotherapy.

[0203] 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 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. In general, dosages vary with age, condition, sex, type of disease, extent 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 in the event of any contraindications. Dosages can vary and can be administered in one or more doses on consecutive days, for one or several days. Guidance can be found in the literature as to appropriate dosages for a given class of pharmaceutical agent.

[0204] As used herein, the terms treatment, treat, or treating refer to a method of reducing the effects of a disease or condition or a symptom of the 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 a symptom of the 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 a disease, condition, or a symptom of the disease or condition.

[0205] As used herein, the terms prevent, prevent and prevent a disease or disorder refer to an action, such as administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, before or at about the same time that a subject begins to show 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 change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

[0206] IL-2 mutants have been developed that are selective for IL2Rαβγ compared to IL2Rβγ (Shanafelt, AB, et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, DJ, et.al., 2002, Curr Pharm Des., 8:2171-83). These mutants have amino acid substitutions that reduce their affinity for IL2RB. Because IL-2 has undetectable affinity for IL2RG, these mutants consequently have a lower affinity for the IL2Rβγ receptor complex and a lower ability to activate IL2Rβγ expressing cells, but retain the ability to bind IL2RA and to bind and activate the IL2Rαβγ receptor complex.

[0207] One of these variants, IL2 / N88R (Bay 50-4798), has been tested in clinical trials as a less toxic form of IL-2 as an immune system stimulator, based on the hypothesis that IL2Rβγ-expressing NK cells are the primary source of toxicity. Bay 50-4798 has been shown to selectively stimulate the proliferation of activated T cells compared to NK cells and has been evaluated in phase I / II clinical trials in cancer patients (Margolin, K., et.al., 2007, Clin Cancer Res., 13:3312-9) and HIV patients (Davey, RT, et.al., 2008, J Interferon Cytokine Res., 28:89-100). These clinical trials demonstrated that Bay 50-4798 was significantly safer and better tolerated than aldesleukin, and also showed that it increased levels of CD4+CD25+ T cells, a cell population enriched for Treg cells. Following these studies, research in this area has further established the identity of Treg cells, demonstrating that Treg cells selectively express IL2Rαβγ (reviewed in Malek, TR, et al., 2010, Immunity, 33:153-65).

[0208] Additionally, variants can be made which selectively alter affinity for the CD25 chain compared to native Il-2.

[0209] IL-2 can be engineered to generate mutants that either bind generally to the IL-2R complex or that specifically bind to the IL-2R α subunit with an affinity different from that of the corresponding wild-type IL-2 or a currently available mutant (designated C125S, in which the cysteine ​​residue at position 125 is replaced with a serine residue).

[0210] Thus, the present invention features a mutant interleukin-2 (IL-2*) polypeptide that includes an amino acid sequence at least 80% identical (e.g., 85%, 87%, 90%, 95%, 97%, 98%, or 99% identical) to wild-type IL-2 and binds to the IL-2 trimeric receptor more highly than the dimeric IL-2 receptor compared to WT IL-2. Typically, the mutant protein also binds to the IL-2 receptor alpha subunit (IL-2Rα) with a higher affinity than wild-type IL-2 binds to IL-2Rα. The amino acid sequence within the mutant IL-2 polypeptide may differ from SEQ ID NO: 1 (UniProtKB Accession No. P60568) by containing (or only containing) one or more amino acid substitutions, which may be considered conservative or non-conservative substitutions. Non-naturally occurring amino acids may also be incorporated. Alternatively, or in addition, the amino acid sequence may differ from SEQ ID NO: 1 (which may be considered the "reference" sequence) by containing, as well as adding and / or deleting, one or more amino acid residues. More specifically, the amino acid sequence may differ from that of SEQ ID NO: 1 by a mutation at at least one of positions 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30, 31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76, 79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 (or a combination thereof) of SEQ ID NO: 1. As mentioned above, as few as one of these positions may be altered, or as many as two, three, four, five, six, seven, eight, nine, ten, or eleven or more (up to and including all) positions may be altered. For example, the amino acid sequence may differ from SEQ ID NO:1 at positions 69 and 74, and may further differ at one or more of positions 30, 35, and 128.The amino acid sequence also includes the following sets of positions: (a) 64, 69, and 74; (b) 69, 74, and 101; (c) 69, 74, and 128; (d) 30, 69, 74, and 103; (e) 49, 69, 73, and 76; (f) 69, 74, 101, and 133; (g) 30, 69, 74, and 128; (h) 69, 74, and 101; (i) 30th, 69th, 74th, 88th, and 99th; (j) 9th, 11th, 35th, 69th, and 74th; (k) 1st, 46th, 49th, 61st, 69th, and 79th; (1) 48th, 68th, 71st, 90th, 103rd, and 114th; (m) 4th, 10th, 11th, 69th, 74th, 88th, and 133rd; (n) 15th, 30th, and 31st. 31st, 35th, 48th, 69th, 74th, and 92nd positions; (O) 30th, 68th, 69th, 71st, 74th, 75th, 76th, and 90th positions; (p) 30th, 31st, 37th, 69th, 73rd, 74th, 79th, and 128th positions; (q) 26th, 29th, 30th, 54th, 67th, 69th, 74th, and 92nd positions; (r) 8th, 1st and (s) one set of positions: 29, 31, 35, 37, 48, 69, 71, 74, 88, and 89. Except for the mutations at these positions, the amino acid sequence of the mutant IL-2 polypeptide may otherwise be identical to SEQ ID NO:1. With respect to specific substitutions, the amino acid sequence may contain the following mutations: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S, N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R, K49E, K54R, E61D, K64R, E67G, E68 D, V69A, N71T, N71A, N71R, A73V, Q74P, S75P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A, or T133N.The nomenclature herein is consistent with that in the scientific literature, where the single letter designation of an amino acid in a wild type or reference sequence is followed by its position in the sequence, followed by the single letter designation of the amino acid it replaces. Thus, A1T represents the substitution of an alanine residue at position 1 with a threonine. Other mutant polypeptides within the scope of the invention include those that contain variants of SEQ ID NO:2 with substitutions at V69 (e.g., A) and Q74 (e.g., P). For example, the amino acid sequence may include the following set of mutations relative to SEQ ID NO:2: (a) K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P, N88D, and S99P, (i) N30S, V69A, Q74P, and I128T, (j) K9T, Q11R, K35R, V69A, and Q74P, (k) A1T, M46L, K49R, E61D, V69A, and H79R, (l) K48E, E68D, N71T, N90H, F103S, and I114V, (m) S4P, T10A, Q11R, V69A, Q74P, N88D, and T133A, (n) E15K, N30S Y31H, K35R, K48E, V69A, Q74P, and I92T, (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and N90H, (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T, (q) N26D, N29S, N30S, K54 The sequences may include one of the following: (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P, and I92T, and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. SEQ ID NO:2 is disclosed in US7569215 and is incorporated herein by reference as an exemplary IL-2 polypeptide sequence that may be used in the present invention.

[0211] As noted above, any of the mutated IL-2 polypeptides disclosed herein can include, or be limited to, the sequences set forth or can otherwise be identical to SEQ ID NO: 1. Additionally, any of the mutated IL-2 polypeptides described herein can optionally include a substitution of the cysteine ​​residue at position 125 with another residue (e.g., serine) and / or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO:1.

[0212] The mutant IL-2 polypeptides disclosed herein have a K for the IL-2Rα subunit of less than about 28 nM. d (e.g., less than about 25 nM, less than about 5 nM, less than about 1 nM, less than about 500 pM, or less than about 100 pM). Specifically, the mutant IL-2 polypeptide may have an affinity equilibrium constant of less than 1.0 nM (e.g., about 0.8 nM, 0.6 nM, 0.4 nM, or 0.2 nM). Affinity may also be expressed as a relative rate of dissociation from the IL-2Rα subunit or IL-2 receptor complex (e.g., a complex expressed on the surface of a cell or otherwise membrane-bound). For example, the mutant IL-2 polypeptide may dissociate from, e.g., IL-2Rα, etc., at a reduced rate compared to a wild-type polypeptide or a therapeutic agent using IL-2, e.g., IL-2*. Alternatively, affinity may be expressed as a relative rate of dissociation from the IL-2Rα subunit or IL-2 receptor complex (e.g., a complex expressed on the surface of a cell or otherwise membrane-bound). * It can be characterized as the time, or average time, that a polypeptide persists on the surface of a cell expressing, for example, IL-2R. * The polypeptide may persist on the receptor for at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 250-fold (or more).

[0213] Disclosed are materials, compositions, and components that can be used in, with, 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 each is specifically contemplated and described herein, even though specific reference to each of the various individual and aggregate combinations and permutations of these compounds may not be expressly disclosed. For example, when a method is disclosed and discussed, and several modifications that can be made to several molecules that comprise the method are discussed, all combinations and permutations of the method, as well as possible modifications, are specifically contemplated unless clearly indicated otherwise. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the disclosure, including, but not limited to, steps in methods that use 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 as specifically contemplated and disclosed.

[0214] Publications cited herein and the material for which they are cited are specifically incorporated herein by reference in their entirety. EXAMPLES

[0215] Below 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.

[0216] Example 1: Detection of IL-2, IL-2 mutant proteins, IL-2Rα and IL-2Rγ in fusion proteins by ELISA IL-2 mutant proteins are detected using a commercially available antibody, e.g., anti-IL-2 monoclonal (JES6-1A12) (BD Pharmingen; San Jose, Calif.). A positive control is used to show whether the monoclonal antibody recognizes the cytokine or the mutant protein. Antibodies against the IL-2Rα and IL-2Rγ chains are also used. Wells of a 96-well plate are coated with PBS containing the antibody (2.5 μg / ml). Wells are blocked with PBS containing 5% nonfat milk with 0.2% Tween® 20 (PBS-M-Tw) and the fusion protein is added for 1-2 hours at 37°C. After washing, anti-IL-2 biotin-labeled antibody, e.g., JES5H4 (BD Pharmingen), is added and binding is detected using Strepavidin HRP (Southern Biotechnology Associates; Birmingham, Ala.). 0.1 M citrate (pH 4.5) and 0.04% H 2 O 2 ELISA plates were prepared by adding 50 μl of O-phenylenediamine (OPD) (Sigma-Aldrich) dissolved in 50 μl / well of 2N H 2 SO 4 The reaction was stopped by adding and the absorbance was read at 490 nm.

[0217] Example 2: 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 ug of protein in 1×PBS (pH 7.4) was cleaved with 1 ug of active MMP9 (Sigma catalog number SAE0078-50 or Enzo catalog BML-SE360) and incubated at room temperature for up to 16 hours. The digested protein is then 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. As shown in Figures 10, 13, 18a, 18b, 24b, 24c, and 27a, complete cleavage of the fusion protein by MMP9 protease is seen.

[0218] Example 3: CTLL-2 Assay CTLL2 cells (ATCC) were suspension seeded at 500,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA) and incubated with serial dilutions of recombinant hIL2 or activatable hIL2 at 37°C, 5% CO 2 Cells were stimulated with 500 mM MMP-9 for 72 hours. The activity of uncleaved and cleaved activatable hIL2 was tested. Cleaved activatable hIL2 was generated by incubation with active MMP9. Cell activity was assessed using the luminescence-based cell viability assay CellTiter-Glo (Promega). The results are shown in Figures 8, 9, and 25.

[0219] Example 4: Protease cleavage of IL-2 / IL-2Rα / IL-2Rγ chimeric polypeptides results in increased antibody accessibility and biologically active IL-2 muteins The IL-2 mutein fusion protein is biochemically characterized before and after cleavage using a protease, e.g., PSA. Immunoblot analysis shows that the fusion protein can be cleaved by PSA, and that after sample treatment with PSA there is an increase in the intensity of the expected low molecular weight cleavage product of approximately 20 kDa that is reactive with anti-IL-2 antibodies. The extent of cleavage depends on the amount of PSA and the incubation time. Interestingly, when the fusion protein is analyzed before and after PSA treatment by ELISA, it is found that the apparent amount of IL-2 increases after PSA cleavage. In this experiment, depending on the construct, there is an approximately two- or four-fold increase in the apparent amount of IL-2 detected using this sandwich ELISA, suggesting that antibody binding is partially prevented with the intact fusion protein. Aliquots of the same samples are also analyzed after PSA treatment using the CTLL-2 cell line, which requires IL-2 for growth and survival, and cell survival can be confirmed using a colorimetric MTT assay. In this assay, the more the supernatant can be diluted, the more biologically active IL-2 it contains, and after PSA cleavage, there is an increase in the amount of biologically active IL-2. The increased amount of IL-2 mutein suggests that after PSA cleavage, there is an increase in the predicted low molecular weight cleavage fragment of approximately 20 kDa that is reactive with anti-IL-2 antibodies, increasing antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutein.

[0220] Example 5. In vivo delivery of protease-activated fusion proteins results in reduced tumor growth The chimeric polypeptides are examined to determine whether they may have biological effects in vivo. These experiments use a system in which tumor cells injected intraperitoneally 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 provides a convenient method for examining the effect of fusion protein therapy on tumor growth, as the fusion protein can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining dissociated omental cells. These experiments may use the Colon38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro. Omental tissue normally expresses relatively low amounts of MMP2 and MMP9, but MMP levels are increased when Colon38 tumors are present in the omentum. This tumor model is used to examine the ability of IL-2 mutant protein fusion proteins to affect tumor growth. Colon38 cells are injected intraperitoneally and allowed to attach and grow for one day before being treated daily with fusion protein intraperitoneally. On day 7, animals are sacrificed and the tumors are examined for tumor growth using flow cytometry and colony formation assays.

[0221] Example 6: Construction of an exemplary activatable IL2 protein that targets CD20 Generation of activatable IL2 domains An IL-2 polypeptide capable of binding to a CD20 polypeptide present on a tumor or 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 nucleic acid sequences of a polypeptide linker. The activatable interleukin plasmid construct, which may have an optional 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 responsive to IFNg stimulation in the presence of a protease.

[0222] 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 more than 90% of B cell non-Hodgkin's lymphomas (NHL). The antigen is not found on hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissues. Therefore, several antibodies, mainly of murine origin, have been described, namely 1F5, 2B8 / C2B8, 2H7, and 1H4.

[0223] Thus, a human or humanized anti-CD20 antibody is used to generate an scFv sequence of the CD20 binding domain of an activatable interleukin protein. DNA sequences encoding the human or humanized VL and VH domains are obtained and the constructs are optionally codon optimized for expression in Homo sapiens cells. The order of appearance of the VL and VH domains in the scFv is changed (i.e., VL-VH or VH-VL orientation) to include the subunits "G4S" (SEQ ID NO: 201) or "G 4 Three copies of "G 4 S) 3 (SEQ ID NO: 204) links the variable domains to create the scFv domain. Anti-CD20 scFv plasmid constructs can have optional Flag, His, or other affinity tags and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20 expressing cells.

[0224] Cloning of a DNA expression construct encoding an activatable IL2 protein An activatable interleukin protein is constructed by combining an anti-CD20 scFv domain and a serum half-life extender (e.g., HSA binding peptide or VH domain) using an activatable IL2 construct with a protease cleavage site domain. For expression of the activatable interleukin protein in CHO cells, the coding sequences of 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 synthesized separately 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-IL2 subunit 1-L2-protease cleavage domain-L3-IL2 subunit 2-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: 205) to facilitate protein secretion and purification, respectively.

[0225] Expression of activatable IL2 protein in stably transfected CHO cells CHO cell expression system (Flp-In®, Life Technologies), CHO-K1 Chinese Hamster Ovary Cell (ATCC, CCL-61) derived cells (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4):1275-81) are used. Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0226] To adapt to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium containing 10% DMSO.

[0227] 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 10 viable cells / mL. After 2-3 weeks of selection, harvest the cell pool that stably expresses 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 the stable cell pool in DMSO-containing medium.

[0228] Activatable IL2 protein is produced by secretion into the cell culture supernatant in a 10-day fed-batch culture of a stably transfected CHO cell line. After 10 days, the 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.

[0229] Protein expression levels and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

[0230] Purification of activatable IL2 protein The activatable IL2 protein is purified from CHO cell culture supernatant in a two-step procedure. In the first step, the construct is subjected to affinity chromatography followed by preparative size-exclusion chromatography (SEC) on Superdex 200 in the second step. The sample is buffer exchanged and concentrated by ultrafiltration to a typical concentration of >1 mg / mL. The purity and homogeneity of the final sample (usually >90%) is assessed by SDS PAGE under reducing and non-reducing conditions followed by immunoblotting using anti-HSA or anti-idiotypic antibodies, respectively, and analytical SEC. The purified protein is stored in equal aliquots at -80°C until use.

[0231] Example 7: Determination of antigen affinity by flow cytometry The activatable interleukin protein of Example 6 was incubated with human CD20 + Cells and cynomolgus monkey CD20 + Their binding affinity to cells is tested.

[0232] 100 μL of serial dilutions of the activatable interleukin protein of Example 6 and CD20 with at least one protease + After washing three times with FACS buffer, the cells are incubated with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotypic antibody in the same buffer for 45 min on ice. After the second washing cycle, the cells are 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 are incubated with anti-His IgG without activatable IL2 protein and then with FITC-conjugated goat anti-mouse IgG antibody. The cells are then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 × 10 4The fluorescence of live cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using Incyte software (Merck Millipore, Schwalbach, Germany). 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 the K D Used to calculate the value.

[0233] CD20 binding and cross-reactivity were determined using human CD20 + Evaluate with tumor cell lines. K D The ratios were calculated using the K D Calculate using the value.

[0234] Example 8: Cytotoxicity Assay The activatable interleukin protein of Example 6 was + Mediating an immune response against target cells is assessed in vitro.

[0235] Fluorescently labeled CD20 +REC-1 cells (Mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMCs of random donors or CB15 T cells (standardized T cell line) as effector cells in the presence of activatable IL2 protein of Example 6 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 by spectrofluorometer. Target cells incubated without activatable IL2 protein of Example 6 and target cells completely lysed by addition of saponin at the end of the incubation are used as negative and positive controls, respectively.

[0236] Based on the measured remaining live target cells, the percentage of specific cell lysis is calculated according to the following formula: [1-(live target (試料) Number of live targets (自然発生的) ) × 100%. Sigmoidal dose-response curves and EC 50 The lysis values ​​obtained for a given antibody concentration are used to calculate a sigmoidal dose-response curve by four-parameter logistic fit analysis using Prism software.

[0237] Example 9: Pharmacokinetics of activatable interleukin proteins The activatable interleukin protein of Example 6 is evaluated in animal studies for half-time loss.

[0238] The activatable IL2 protein is administered to cynomolgus monkeys as a 0.5 mg / kg bolus injection into the saphenous vein. Another group of cynomolgus monkeys receives an IL2 construct of comparable size but lacking a serum half-life extender. A third and fourth group receive an IL2 construct with a serum half-life extender, and a cytokine with CD20 and a serum half-life extender, respectively, both of comparable size to the activatable interleukin protein. Each test group consists of five monkeys. Serum samples are taken at the indicated time points and serially diluted, and protein concentrations are determined using a binding ELISA against CD20.

[0239] 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-phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D / V(α-k21) / (α-β), 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.

[0240] Data Analysis: Graphs of concentration versus time profiles 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 graphed. 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.

[0241] The activatable interleukin proteins of Example 6 are expected to have improved pharmacokinetic parameters, such as increased elimination half-times, compared to proteins lacking the serum half-life extending element.

[0242] Example 10: Xenograft tumor model The activatable IL2 protein of Example 6 is evaluated in a xenograft model.

[0243] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and given 4 × 10 6 Ramos RA1 cells were inoculated subcutaneously into the right dorsal flank. 3 Once the immunization rate reaches 1.5×10, animals are assigned to three treatment groups: Group 2 and Group 3 (8 animals each) with 1.5×10 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 Example 6 for a total of nine intravenous doses (once a day for nine days). Groups 1 and 2 are treated with vehicle only. Body weight and tumor volume are measured for 30 days.

[0244] Animals treated with the activatable interleukin proteins of Example 6 are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0245] 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 present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0246] Example 11: Mouse IFNg WEHI cell survival assay WEHI279 cells (ATCC) were seeded at 25,000 cells / well in suspension in medium with or without 1.5% human serum albumin (HSA) and incubated with serial dilutions of recombinant or inducible mIFNg at 37°C and 5% CO 2 Cells were stimulated with 500 mM MMP-9 for 72 hours. The activity of uncleaved and truncated inducible mIFNg was tested. Truncated inducible mIFNg was generated by incubation with active MMP9. Cell viability was assessed using a CellTiter-Glo (Promega) luminescence-based cell viability assay. The EC50 values ​​of the truncated inducible mIFNg molecules were at least 100-fold more potent than the uncleaved inducible mIFNg molecules. As shown in FIG. 16, higher inducibility was seen in assays in which human serum albumin was included in the medium.

[0247] Example 12: Mouse IFNg B16 reporter cell assay B16-Blue IFNg cells (InvivoGen) were seeded at a concentration of 75,000 cells / well in medium with or without 1.5% human serum albumin (HSA) and incubated with serial dilutions of recombinant or inducible mIFNg at 37°C and 5% CO. 2 The cells were stimulated with MMP9 for 24 hours. The activity of uncleaved and truncated inducible mIFNg was tested. The truncated inducible mIFNg was generated by incubation with active MMP9. The supernatant was collected and incubated with QUANTI-Blue reagent (InvivoGen) for 2 hours at 37°C, and SEAP activation was evaluated by measuring the absorbance at 620 nm. The results are shown in Figures 17, 19, 22, 23, and 28. The same experiment was repeated for IFNa fusion protein using B16-Blue IFNa / B cells. The EC50 value of the truncated inducible mIFNa molecule was at least 100-fold more potent than the uncleaved inducible mIFNa molecule.

[0248] Example 13. In vivo delivery of protease-activated fusion proteins results in reduced tumor growth The chimeric polypeptides are examined to determine whether they may have biological effects in vivo. These experiments use a system in which tumor cells injected intraperitoneally 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 provides a convenient method for examining the effect of fusion protein therapy on tumor growth, as the fusion protein can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining dissociated omental cells. These experiments may use the Colon38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro. Omental tissue normally expresses relatively low amounts of MMP2 and MMP9, but MMP levels are increased when Colon38 tumors are present in the omentum. This tumor model is used to examine the ability of IFN fusion proteins to affect tumor growth. Colon38 cells are injected intraperitoneally and allowed to attach and grow for one day before being treated daily with fusion protein intraperitoneally. On day 7, animals are sacrificed and the tumors are examined for tumor growth using flow cytometry and colony formation assays.

[0249] Example 13b: The chimeric polypeptide was examined to determine its biological effect in vivo. The MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. This tumor model was used to examine the ability of IFNγ fusion proteins to affect tumor growth. MC38 cells were injected subcutaneously and allowed to grow for 10-14 days, then treated intraperitoneally with fusion proteins at the levels shown in Figure 21 twice a week for a total of four doses. As a comparative control, wild-type mIFNγ was administered twice a day at the indicated dose levels on a 5 day on / 2 day off schedule (total of 10 doses) for two weeks. Tumor growth and body weight were monitored approximately twice a week for two weeks.

[0250] Example 14: Construction of an exemplary IFNg protein that targets CD20 Generation of activatable cytokine domains An IFNg 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 nucleic acid sequences of a polypeptide linker. An activatable IFNg plasmid construct can have an optional 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 responsive to IFNg stimulation in the presence of a protease.

[0251] 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 more than 90% of B cell non-Hodgkin's lymphomas (NHL). The antigen is not found on hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissues. Therefore, several antibodies, mainly of murine origin, have been described, namely 1F5, 2B8 / C2B8, 2H7, and 1H4.

[0252] Thus, a human or humanized anti-CD20 antibody is used to generate an scFv sequence of the CD20 binding domain of an activatable IFNg protein. DNA sequences encoding the human or humanized VL and VH domains are obtained and the constructs are optionally codon optimized for expression in Homo sapiens cells. The order of appearance of the VL and VH domains in the scFv is changed (i.e., VL-VH or VH-VL orientation) to include the subunits "G4S" (SEQ ID NO: 201) or "G 4 Three copies of "G 4 S) 3(SEQ ID NO: 204) links the variable domains to create the scFv domain. Anti-CD20 scFv plasmid constructs can have optional Flag, His, or other affinity tags and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20 expressing cells.

[0253] Cloning of a DNA expression construct encoding an activatable IFNg protein An activatable IFNg protein is constructed using an activatable IFNg construct with a protease cleavage site domain in combination with an anti-CD20 scFv domain and a serum half-life extender (e.g., HSA binding peptide or VH domain), with the domains organized as shown in FIG. 14. For expression of the activatable IFNg protein in CHO cells, the coding sequences of all protein domains are cloned into a mammalian expression vector system. Briefly, gene sequences encoding the activatable IFNg domain, serum half-life extender, and CD20 binding domain are synthesized separately 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-IFNg subunit 1-L2-protease cleavage domain-L3-IFNg subunit 2-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: 205) to facilitate protein secretion and purification, respectively. Expression of activatable IFNg protein in stably transfected CHO cells

[0254] CHO cell expression system (Flp-In®, Life Technologies), CHO-K1 Chinese Hamster Ovary Cell (ATCC, CCL-61) derived cells (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4):1275-81) are used. Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0255] To adapt to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium containing 10% DMSO.

[0256] Recombinant CHO cell lines stably expressing secreted, activatable IFNg protein are generated by transfection of suspension-adapted cells. Upon selection with the antibiotic hygromycin B, viable cell density is measured twice a week and cells are centrifuged to a maximum density of 0.1 × 10 6 Resuspend in fresh selection medium at 10 viable cells / mL. After 2-3 weeks of selection, harvest the cell pool that stably expresses the activatable IFNg 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 the stable cell pool in DMSO-containing medium.

[0257] Activatable IFNg protein is produced by secretion into cell culture supernatant in a 10-day fed-batch culture of a stably transfected CHO cell line. 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, cell culture supernatant is removed by centrifugation and vacuum filtered before further use.

[0258] Protein expression levels and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

[0259] Purification of activatable IFNg protein Activatable IFNg protein is purified from CHO cell culture supernatant in a two-step procedure. In the first step, the construct is subjected to affinity chromatography followed by preparative size-exclusion chromatography (SEC) on Superdex 200 in the second step. 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, respectively, and analytical SEC. The purified protein is stored at -80°C in equal aliquots until use.

[0260] Example 15: Determination of antigen affinity by flow cytometry The activatable IFNg protein of Example 14 was incubated with human CD20 + Cells and cynomolgus monkey CD20 + Their binding affinity to cells is tested.

[0261] 100 μL of serial dilutions of the activatable IFNg protein of Example 14 and at least one protease together with CD20 + After washing three times with FACS buffer, the cells are incubated with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotypic antibody in the same buffer for 45 min on ice. After the second washing cycle, the cells are 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 are incubated with anti-His IgG without activatable IFNg protein and then with FITC-conjugated goat anti-mouse IgG antibody. The cells are then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 × 10 4The fluorescence of live cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using Incyte software (Merck Millipore, Schwalbach, Germany). 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 the K D Used to calculate the value.

[0262] CD20 binding and cross-reactivity were determined using human CD20 + Evaluate with tumor cell lines. K D The ratios were calculated using the K D Calculate using the value.

[0263] Example 16: Cytotoxicity Assay The activatable IFNg protein of Example 14 was + Mediating an immune response against target cells is assessed in vitro.

[0264] 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 (standardized T cell line) as effector cells in the presence of the activatable IFNg protein of Example 14 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 by spectrofluorometer. Target cells incubated without the activatable IFNg protein of Example 14 and target cells completely lysed by addition of saponin at the end of the incubation are used as negative and positive controls, respectively.

[0265] Based on the measured remaining live target cells, calculate the percentage of specific cell lysis according to the following formula: [1-(live target (試料) Number of live targets (自然発生的) ) × 100%. Sigmoidal dose-response curves and EC 50 The lysis values ​​obtained for a given antibody concentration are used to calculate a sigmoidal dose-response curve by four-parameter logistic fit analysis using Prism software.

[0266] Example 17: Pharmacokinetics of activatable IFNg proteins The activatable IFNg protein of Example 14 is evaluated in animal studies for half-time loss.

[0267] The activatable IFNg 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 extender. A third and fourth group receive a cytokine with a serum half-life extender and a cytokine with CD20 and a serum half-life extender, respectively, both of comparable size to the activatable IFNg protein. Each test group consists of five monkeys. Serum samples are taken at the indicated time points and serially diluted, and protein concentrations are determined using a binding ELISA against CD20.

[0268] 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-phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D / V(α-k21) / (α-β), 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.

[0269] Data Analysis: Graphs of concentration versus time profiles 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 graphed. 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.

[0270] The activatable IFNg protein of Example 14 is expected to have improved pharmacokinetic parameters, such as increased elimination half-times, compared to proteins lacking the serum half-life extending element.

[0271] Example 18: Xenograft tumor model The activatable IFNg protein of Example 14 is evaluated in a xenograft model.

[0272] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and given 4 × 10 6 Ramos RA1 cells were inoculated subcutaneously into the right dorsal flank. 3 Once the immunization rate reaches 1.5×10, animals are assigned to three treatment groups: Group 2 and Group 3 (8 animals each) with 1.5×10 7of activated human T cells are injected intraperitoneally. Three days later, animals in group 3 are subsequently treated with 50 μg of the activatable IFNg protein of Example 14 for a total of nine intravenous doses (once a day for nine days). Groups 1 and 2 are treated with vehicle only. Body weights and tumor volumes are measured for 30 days.

[0273] Animals treated with the activatable IFNg protein of Example 14 are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0274] 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 present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby.

[0275] Example 19: HEK Blue Assay HEK-Blue IL12 cells (InvivoGen) were suspension seeded at 250,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA) and incubated with serial dilutions of recombinant hIL12, chimeric IL12 (mouse p35 / human p40) or activatable hIL12 at 37°C and 5% CO 2 The cells were stimulated with 500 mM MMP-9 for 24 hours. The activity of uncleaved and truncated activatable hIL12 was tested. Truncated inducible 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-based assay. The results are shown in Figure 11, Figure 12, Figure 15, and Figure 26.

[0276] HEK-Blue IL2 cells (InvivoGen) were suspension-seeded at 50,000 cells / well in medium with or without 15–40 mg / ml human serum albumin (HSA) and incubated with serial dilutions of recombinant or activatable hIL2 at 37°C and 5% CO. 2 The cells were stimulated with 100 mM MMP-9 for 24 hours. The activity of uncleaved and truncated activatable hIL2 was tested. Truncated inducible hIL2 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-based assay. The results are shown in Figures 24a-d.

[0277] Example 20: Splenocyte T-Blast Assay T-Blasts were derived from mouse splenocytes by incubation with PHA for 6 days and recombinant hIL12 for 24 hours. Tblasts were then suspended and seeded at 200,000 cells / well in medium with or without 40 mg / ml human serum albumin (HSA) and incubated at 37°C in 5% CO with serial dilutions of recombinant hIL12 or chimeric IL12 (mouse p35 / human p40) or mouse IL12. 2 Cells were stimulated with IL-12 for 72 hours. The activity of uncleaved and truncated forms of IL-12 was tested. Truncated inducible hIL-12 was generated by incubation with active MMP-9. IL-12 activity was assessed by downstream quantification of IFN-gamma production using mIFN-gamma alphaLISA.

[0278] Example 21: In vivo delivery of protease-activated fusion proteins results in reduced tumor growth The chimeric polypeptides are examined to determine whether they may have 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 provides a convenient method for examining the effect of fusion protein therapy on tumor growth, as the fusion protein can be delivered multiple times intraperitoneally and tumor growth can be analyzed by examining dissociated omental cells. These experiments may use the Colon38 cell line, a rapidly growing tumor cell line that expresses both MMP2 and MMP9 in vitro. Omental tissue normally expresses relatively low amounts of MMP2 and MMP9, but MMP levels are increased when Colon38 tumors are present in the omentum. This tumor model is used to examine the ability of IL-2 mutant protein fusion proteins to affect tumor growth. Colon38 cells are injected intraperitoneally and allowed to attach and grow for one day before being treated daily with fusion protein intraperitoneally. On day 7, animals are sacrificed and the tumors are examined for tumor growth using flow cytometry and colony formation assays.

[0279] Example 22: Construction of an exemplary activatable interleukin protein that targets CD20 Generation of activatable interleukin domains The human IL-12p35 chain standard sequence is Uniprot Accession No. P29459. The human IL-12p40 chain standard sequence is Uniprot Accession No. 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 generated as follows: A nucleic acid is generated to contain (1) a nucleic acid sequence encoding an IFNg polypeptide sequence and (2) one or more nucleic acid sequences of a polypeptide linker. The activatable interleukin plasmid construct can have an optional Flag, His, or other affinity tag, and is electroporated into HEK293 or other suitable human or mammalian cell 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.

[0280] 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 more than 90% of B cell non-Hodgkin's lymphomas (NHL). The antigen is not found on hematopoietic stem cells, activated B lymphocytes (plasma cells) and normal tissues. Therefore, several antibodies, mainly of murine origin, have been described, namely 1F5, 2B8 / C2B8, 2H7, and 1H4.

[0281] Thus, a human or humanized anti-CD20 antibody is used to generate an scFv sequence of the CD20 binding domain of an activatable interleukin protein. DNA sequences encoding the human or humanized VL and VH domains are obtained and the constructs are optionally codon optimized for expression in Homo sapiens cells. The order of appearance of the VL and VH domains in the scFv is changed (i.e., VL-VH or VH-VL orientation) to include the subunits "G4S" (SEQ ID NO: 201) or "G 4Three copies of "G 4 S) 3 (SEQ ID NO: 204) links the variable domains to create the scFv domain. Anti-CD20 scFv plasmid constructs can have optional Flag, His, or other affinity tags and are electroporated into HEK293 or other suitable human or mammalian cell lines and purified. Validation assays include binding analysis by FACS, kinetic analysis using Proteon, and staining of CD20 expressing cells.

[0282] Cloning of DNA expression constructs encoding activatable interleukin proteins. An activatable interleukin protein is constructed in combination with an anti-CD20 scFv domain and a serum half-life extender (e.g., HSA-binding peptide or 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 of 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 synthesized separately 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: 205) to facilitate protein secretion and purification, respectively.

[0283] Expression of activatable interleukin proteins in stably transfected CHO cells CHO cell expression system (Flp-In®, Life Technologies), CHO-K1 Chinese Hamster Ovary Cell (ATCC, CCL-61) derived cells (Kao and Puck, Proc. Natl. Acad Sci USA 1968;60(4):1275-81) are used. Adherent cells are subcultured according to the standard cell culture protocol provided by Life Technologies.

[0284] To adapt to growth in suspension, cells are detached from tissue culture flasks and placed in serum-free medium. Suspension-adapted cells are cryopreserved in medium containing 10% DMSO.

[0285] 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 10 viable cells / mL. After 2-3 weeks of selection, harvest the cell pool that stably expresses 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 the stable cell pool in DMSO-containing medium.

[0286] Activatable interleukin proteins are produced by secretion into cell culture supernatant in a 10-day fed-batch culture of a stably transfected CHO cell line. After 10 days, cell culture supernatant is harvested, typically 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, cell culture supernatant is removed by centrifugation and vacuum filtered before further use.

[0287] Protein expression levels and product integrity in cell culture supernatants are analyzed by SDS-PAGE.

[0288] Purification of activatable interleukin proteins The 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, respectively, and analytical SEC. The purified proteins are stored in equal aliquots at -80°C until use.

[0289] Example 23: Determination of antigen affinity by flow cytometry The activatable interleukin protein of Example 22 was incubated with human CD20 + Cells and cynomolgus monkey CD20 + Their binding affinity to cells is tested.

[0290] 100 μL of serial dilutions of the activatable interleukin protein of Example 22 and CD20 with at least one protease + After washing three times with FACS buffer, the cells are incubated with 0.1 mL of 10 μg / mL mouse monoclonal anti-idiotypic antibody in the same buffer for 45 min on ice. After the second washing cycle, the cells are 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 are incubated with anti-His IgG without activatable interleukin protein and then with FITC-conjugated goat anti-mouse IgG antibody. The cells are then washed again and resuspended in 0.2 mL of FACS buffer containing 2 μg / mL propidium iodide (PI) to exclude dead cells. 1 × 10 4The fluorescence of live cells is measured using a Beckman-Coulter FC500 MPL flow cytometer using MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore Guava EasyCyte flow cytometer using Incyte software (Merck Millipore, Schwalbach, Germany). 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). California USA) using the equation for one-site binding (hyperbola) D Used to calculate the value.

[0291] CD20 binding and cross-reactivity were determined using human CD20 + Evaluate with tumor cell lines. K D The ratios were calculated using the K D Calculate using the value.

[0292] Example 24: Cytotoxicity Assay The activatable interleukin protein of Example 22 was + Mediating an immune response against target cells is assessed in vitro.

[0293] 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 (standardized T cell line) as effector cells in the presence of the activatable interleukin protein of Example 22 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 by spectrofluorometer. Target cells incubated without the activatable interleukin protein of Example 22 and target cells completely lysed by addition of saponin at the end of the incubation are used as negative and positive controls, respectively.

[0294] Based on the measured remaining live target cells, the percentage of specific cell lysis is calculated according to the following formula: [1-(live target (試料) Number of live targets (自然発生的) ) × 100%. Sigmoidal dose-response curves and EC 50 The lysis values ​​obtained for a given antibody concentration are used to calculate a sigmoidal dose-response curve by four-parameter logistic fit analysis using Prism software.

[0295] Example 25: Pharmacokinetics of activatable interleukin proteins The activatable interleukin protein of Example 22 is evaluated in animal studies for half-time loss.

[0296] 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 extender. A third and fourth group receive a cytokine with a serum half-life extender and a cytokine with CD20 and a serum half-life extender, respectively, both of comparable size to the activatable interleukin protein. Each test group consists of five monkeys. Serum samples are taken at the indicated time points and serially diluted, and protein concentrations are determined using a binding ELISA against CD20.

[0297] 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-phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A=D / V(α-k21) / (α-β), 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.

[0298] Data Analysis: Graphs of concentration versus time profiles 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 graphed. 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.

[0299] The activatable interleukin proteins of Example 22 are expected to have improved pharmacokinetic parameters, such as increased elimination half-times, compared to proteins lacking the serum half-life extending element.

[0300] Example 26: Xenograft tumor model The activatable interleukin protein of Example 22 is evaluated in a xenograft model.

[0301] Female immunodeficient NOD / scid mice were sublethally irradiated (2 Gy) and given 4 × 10 6 Ramos RA1 cells were inoculated subcutaneously into the right dorsal flank. 3 Once the immunization rate reaches 1.5×10, animals are assigned to three treatment groups: Group 2 and Group 3 (8 animals each) with 1.5×10 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 Example 22 for a total of nine intravenous doses (once a day for nine days). Groups 1 and 2 are treated with vehicle only. Body weight and tumor volume are measured for 30 days.

[0302] Animals treated with the activatable interleukin protein of Example 22 are expected to have a statistically significant delay in tumor growth compared to the respective vehicle-treated control groups.

[0303] 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 present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in the practice of the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims, and their equivalents, be covered thereby. Example 27: MC38 Experiments We used the MC38 cell line, a rapidly growing colon adenocarcinoma cell line that expresses MMP9 in vitro. This tumor model was used to examine the ability of the fusion protein to affect tumor growth. Example 27a: MC38 IL-2POC Drugs and Treatment: [Table 4-1] [Table 4-2] Example 27b: MC38 IL-2 Drugs and Treatment: [Table 5-1] [Table 5-2] The results are shown in Figures 31a-c and 32a-c, and the survival curves are shown in Figures 34a-d. Example 27c: MC38 IFNa and IL-12 Drugs and Treatment: [Table 6-1] [Table 6-2] Example 27e: MC38 rechallenge Cured mice from Example 27b (ACP16 treated) were re-challenged with tumor implants to determine whether anti-tumor memory had been established from the initial treatment. Drugs and Treatment: [Table 7-1] [Table 7-2]

[0304] Example 28. 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 an 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 operatively linked to the active moiety through a protease-cleavable linker, and the active moiety is operatively linked to a targeting domain (here an anti-epidermal growth factor receptor (EGFR) dAb or an anti-prostate specific membrane antigen (PSMA) dAb) through 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 cytokine desired 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 and inhibits the cytokine or a functional fragment or mutein thereof can be prepared using the methods described and exemplified herein. An anti-serum albumin dAb that binds to a desired cytokine or a functional fragment or mutein thereof and inhibits its activity can provide both steric masking of the cytokine (due to the proximity of the cytokine to the serum albumin to which it is bound) and specific masking of the cytokine (due to binding via a non-CDR loop (e.g., C-loop) to the cytokine). Anti-serum albumin dAbs that bind to a desired cytokine or a functional fragment or mutein thereof 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.Selection can use any suitable method, such as phage display. For example, an exemplary anti-serum albumin dab that can be used has the sequence below, where the amino acid sequence in the C-loop (bold 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]

[0305] A. Protease activation of ProTriTAC results in greatly enhanced activity in vitro 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.

[0306] For ELISA, soluble ProTriTAC protein at the indicated concentrations 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 using PBS containing 0.05% Tween-20, and detected using the non-competitive anti-CD3 idiotypic monoclonal antibody 11D3, followed by peroxidase-conjugated secondary antibody and TMB-ELISA substrate solution (Thermo Fisher).

[0307] 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 h at 4° C. Plates were washed with 2% fetal bovine serum in PBS and detected using AlexaFluor 647-labeled noncompetitive anti-CD3 idiotypic monoclonal antibody 11D3, and data were analyzed using FlowJo 10 (FlowJo, LLC).

[0308] 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 h at 37° C. The HCT116 target cell line was stably transfected with a luciferase reporter gene to allow measurement of specific T cell-mediated cell killing by ONE-Glo (Promega).

[0309] 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, 5x106 HCT116 cells were mixed with 2.5x106 expanded T cells per mouse on day 0. ProTriTAC administration began the following day and was performed by intraperitoneal injection on a once daily x10 schedule. At the indicated times, tumor volume measurements were obtained using caliper measurements and calculated using the formula V=(length x width x width) / 2.

[0310] C. Expression, purification and stability of exemplary ProTriTAC trispecific molecules Protein production Sequences encoding the inducible fusion protein molecules were cloned into the mammalian expression vector pcDNA 3.4 (Invitrogen) after the leader sequence and before the 6x histidine tag (SEQ ID NO: 205). Expi293F cells (Life Technologies A14527) were maintained in suspension in Optimum Growth Flasks (Thomson) at 0.2-8x1e6 cells / ml in Expi 293 medium. Expi293 cells were transfected with purified plasmid DNA according to the Expi293 Expression System Kit (Life Technologies, A14635) protocol and maintained for 4-6 days after transfection. Alternatively, sequences encoding the fusion protein molecules were cloned into the mammalian expression vector pDEF38 (CMC ICOS) and transfected into CHO-DG44dhfr- cells to generate stable pools and were 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 in additive-containing neutral buffer with fraction pooling. Final purity was assessed by SDS-PAGE and ELISA using an Acquity BEH SEC 200 Analytical SEC using a 1.7u 4.6 x 150mm column (Waters Corporation) was used to evaluate the fusion protein, which was split into aqueous / organic mobile phases with neutral pH additives on a 1290 LC System and peaks were integrated with Chemstation CDS software (Agilent). The fusion protein purified from CHO host cells is shown on SDS-PAGE below.

[0311] Stability assessment The purified fusion protein in the two formulations was further divided into equal portions into sterile tubes and stressed by five freeze-thaw cycles consisting of one at -80°C and room temperature for >1 hour, or by incubation at 37°C for 1 week. The 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.

[0312] The results show that ProTriTAC was produced in yields comparable to regular TriTAC from CHO stable pools, and the protein was stable after repeated freeze-thaw cycles and after one week at 37°C.

[0313] D. Demonstration of Functional Masking and Stability of ProTriTAC In Vivo in a 3-Week Cynomolgus Monkey Pharmacokinetic Study A single dose of PSMA-directed ProTriTAC (SEQ ID NO: C1872), uncleavable ProTriTAC (SEQ ID NO: 187), unmasked / uncleavable TriTAC (SEQ ID NO: 190), and protease-activated ProTriTAC mimicking the active drug (SEQ ID NO: 188) was administered to cynomolgus monkeys at 0.1 mg / kg by intravenous injection. Plasma samples were collected at the indicated time points. ProTriTAC concentrations were determined using a ligand binding assay with biotinylated recombinant human PSMA (R&D systems) and 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 a non-compartmental method consistent with the intravenous bolus administration route.

[0314] To calculate the in vivo prodrug conversion rate, the circulating blood concentration of active drug is calculated using the following simultaneous differential equations [where P is the concentration of prodrug, A is the concentration of 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

[0315] Prodrugs, active drugs, and non-cleavable prodrug controls (k c,NCLV ) was empirically determined in cynomolgus monkeys. To estimate the rate of circulating prodrug activation, it was assumed that the difference in clearance rate between cleavable and non-cleavable prodrugs arises solely from non-specific activation in the circulation. Thus, 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

[0316] The initial concentration of circulating prodrug was determined empirically and the initial concentration of active drug was assumed to be zero.

[0317] Results and Discussion The results of Example 28 show that fusion proteins containing a polypeptide with a desired therapeutic activity, such as a cytokine or a 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 show that fusion proteins of this type can be administered as inactive proteins that are activated upon protease cleavage at the desired location of therapeutic activity, such as a tumor.

[0318] The amino acid sequences of the fusion proteins used in Example 28 are shown in SEQ ID NOs: 183-190.

[0319] 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] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

Table 3-8

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 8-10

Table 8-11

Table 8-12

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 8-22

Table 8-23

Table 8-24

Table 8-25

Table 8-26

Table 8-27

Table 8-28

Table 8-29

Table 8-30

Table 8-31

Table 8-32

Table 8-33

[0320] Incorporation by Reference The entire disclosures of all patent and non-patent publications cited herein are each incorporated by reference in their entirety for all purposes.

[0321] 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 illustrated herein should not be considered in a limiting sense. The subject matter of the present disclosure includes all novel and non-obvious combinations and combinations of the various elements, features, functions, and / or properties disclosed herein. The following claims specifically point out certain combinations and combinations that are considered to be novel and non-obvious. 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 are also considered to be within the subject matter of the present disclosure, regardless of whether they are directed to different or the same invention, and whether they are broader, narrower, equal, or different in scope compared to the scope of the original claims. The present invention provides, for example, the following items. (Item 1) a) cytokine polypeptide [A]; b) a cytokine blocking moiety [D], and c) a protease-cleavable polypeptide linker [L], The cytokine polypeptide and the cytokine blocking moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated cytokine receptor activating activity, the cytokine receptor activating activity of the fusion polypeptide being at least about 10-fold less than the cytokine receptor activating activity of a polypeptide containing the cytokine polypeptide produced by cleavage of the protease-cleavable linker. (Item 2) 2. The fusion polypeptide according to item 1, wherein the cytokine polypeptide is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23, TGFβ, IFNα, IFNβ, IFNγ, TNF, TGF beta, CXCL10, CCL19, CCL20, CCL21 and active fragments thereof. (Item 3) 3. The fusion polypeptide according to item 1 or 2, wherein the cytokine polypeptide is a cytokine polypeptide with a short half-life. (Item 4) The fusion polypeptide according to any one of items 1 to 3, wherein the protease-cleavable linker polypeptide comprises a sequence capable of being cleaved by a protease independently selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloprotease (MMP), ADAM, FAP, cathepsin L, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease. (Item 5) 5. The fusion polypeptide of any one of claims 1 to 4, wherein the protease-cleavable polypeptides independently comprise 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 comprises cleavage sites for two or more different proteases. (Item 6) 6. The fusion polypeptide according to any one of items 1 to 5, wherein the cytokine blocking moiety is also a half-life extending element. (Item 7) 7. The fusion polypeptide according to any one of items 1 to 6, wherein the cytokine-blocking moiety sterically blocks the agonist activity of the cytokine. (Item 8) 8. The fusion polypeptide of item 6 or 7, wherein the cytokine blocking moiety is human serum albumin, an antigen-binding protein, or an antigen-binding polypeptide that binds to human serum albumin or a fragment thereof. (Item 9) 9. The fusion polypeptide according to any one of items 1 to 8, wherein the cytokine is freely dissociated from the cytokine blocking moiety after the protease-cleavable polypeptide linker is cleaved by a protease. (Item 10) 10. The fusion polypeptide according to any one of items 1 to 9, wherein the cytokine blocking moiety inhibits the cytokine polypeptide from activating its cognate receptor. (Item 11) 11. The fusion polypeptide according to any of items 1 to 10, wherein the fusion polypeptide binds to the cognate receptor of the cytokine polypeptide prior to cleavage by the protease-cleavable linker. (Item 12) 12. The fusion polypeptide of any one of items 1 to 11, further comprising at least one half-life extension element. (Item 13) 13. The fusion polypeptide according to any one of items 1 to 12, wherein the cytokine blocking moiety is non-covalently associated with the cytokine polypeptide, independent of a peptide bond of the linker. (Item 14) 14. The fusion polypeptide of claim 13, wherein the non-covalent association is pH dependent. (Item 15) 13. The fusion polypeptide of item 12, wherein the half-life extending element sterically inhibits or blocks activation and / or binding of the cytokine polypeptide to its cognate receptor. (Item 16) 13. The fusion polypeptide of item 12, wherein the half-life extending element is human serum albumin, human IgG, humanized IgG, scFv, Fab, sdAb or a fragment thereof. (Item 17) 2. The fusion polypeptide of claim 1, wherein the cytokine blocking moiety comprises a ligand binding domain, a single domain antibody or scFv that binds to the cytokine polypeptide, or an antibody or antibody fragment selected from a single domain antibody and an scFv that binds to a receptor for the cytokine polypeptide. (Item 18) 18. The fusion polypeptide according to any one of items 1 to 17, wherein the cytokine receptor activating activity is determined using a standard in vitro receptor activation assay and equal amounts of the cytokine polypeptide and fusion protein on a molar basis. (Item 19) 19. The fusion polypeptide according to any one of items 1 to 18, wherein the cytokine loses contact with the cytokine blocking moiety and / or half-life extension element after the protease-cleavable sequence is cleaved by a protease. (Item 20) The fusion polypeptide is characterized in that at least one of [A], [D], and [L] is selected from the following: a) [A]-[L]-[D], b) [D]-[L]-[A], c)[D]-[L]-[A]-[L]-[D]-[L]-[A], d)[A]-[L]-[D]-[L]-[A]-[L]-[D], e) [D]-[L]-[A]-[L]-[D], f)[D]-[L]-[A]-[L]-[A]-[L]-[D], 20. The fusion polypeptide according to any one of items 1 to 19, comprising the fusion polypeptide in any one of the following orientations: g) [D]-[L]-[A]-[L]-[D]-[L]-[A]-[L]-[D]; and h) [D]-[L]-[A]-[A]-[L]-[D]. (Item 21) 21. The fusion polypeptide according to any one of items 1 to 20, wherein the fusion polypeptide comprises two cytokine peptides. (Item 22) formula [A]-[L1]-[D]-[L2]-[A]-[L2]-[D] [In the formula, A is a cytokine polypeptide, L1 and L2 are each independently a protease-cleavable polypeptide linker; D is a cytokine blocking moiety that can optionally extend serum half-life; 2. The fusion polypeptide according to item 1, wherein L1 is a substrate for a first protease and L2 is a substrate for a second protease. (Item 23) 13. The fusion polypeptide of any one of items 12, wherein the half-life extension is one half-life extension, two half-life extensions, or three half-life extensions. (Item 24) 24. The fusion polypeptide according to any one of items 1 to 23, comprising one protease-cleavable linker, two protease-cleavable linkers, three protease-cleavable linkers, four protease-cleavable linkers, five protease-cleavable linkers, six protease-cleavable linkers, or seven protease-cleavable linkers. (Item 25) 25. The fusion polypeptide according to any one of items 1 to 24, further comprising a tumor-specific antigen-binding peptide. (Item 26) 26. The fusion polypeptide of claim 25, wherein the tumor-specific antigen-binding peptide is operably linked to the cytokine polypeptide by a non-cleavable linker. (Item 27) 26. The fusion polypeptide of claim 25, wherein the tumor-specific antigen-binding peptide is operably linked to the cytokine polypeptide by a cleavable linker. (Item 28) 28. The fusion polypeptide according to any one of items 1 to 27, wherein the cytokine polypeptide has a serum half-life comparable to that of a corresponding, naturally occurring cytokine. (Item 29) 28. The fusion polypeptide according to any one of items 1 to 27, wherein the serum half-life of the polypeptide containing the cytokine polypeptide produced by cleavage of the fusion protein is equivalent to that of a corresponding naturally occurring cytokine. (Item 30) 28. The fusion polypeptide according to any one of items 1 to 27, wherein the cytokine polypeptide is a mutant protein that has a short serum half-life and is rapidly eliminated after cleavage of the fusion polypeptide. (Item 31) a) interferon polypeptide [A], b) an interferon-blocking moiety [D], and c) a protease-cleavable polypeptide linker [L], The fusion polypeptide, wherein the interferon polypeptide and the interferon blocking moiety are operably linked by the protease-cleavable polypeptide linker, and the fusion polypeptide has attenuated interferon receptor activating activity, the interferon receptor activating activity of the fusion polypeptide being at least about 10-fold less than the interferon receptor activating activity of the interferon polypeptide produced by cleavage of the protease-cleavable linker. (Item 32) formula [D]-[L1]-[A]-[L2]-[D] [In the formula, A is an interferon alpha (IFNα) polypeptide; L1 and L2 are each independently a protease-cleavable polypeptide linker; D is an IFNα blocking moiety that also extends in vivo half-life; 32. The fusion polypeptide of claim 31, wherein the interferon alpha polypeptide and the interferon alpha blocking moiety are operably linked by the protease-cleavable polypeptide linker, the fusion polypeptide has attenuated interferon alpha receptor activating activity, the interferon alpha receptor activating activity of the fusion polypeptide being at least about 10-fold lower than the interferon receptor activating activity of the interferon alpha polypeptide produced by cleavage of the protease-cleavable linker, and, upon cleavage of both linkers, the in vivo half-life of the interferon alpha polypeptide is substantially similar to the in vivo half-life of naturally occurring interferon alpha. (Item 33) formula [D]-[L1]-[A]-[L2]-[D]-[L3]-[A]-[L4]-[D], [in the formula, A is an interferon gamma (IFNγ) polypeptide; L1, L2, L3, and L4 are each independently a protease-cleavable polypeptide linker; D is an IFNγ blocking moiety capable of extending serum half-life; 32. The fusion polypeptide of claim 31, wherein the interferon gamma polypeptide and the interferon gamma blocking moiety are operably linked by the protease-cleavable polypeptide linker, the fusion polypeptide has attenuated interferon gamma receptor activating activity, the interferon gamma receptor activating activity of the fusion polypeptide being at least about 10-fold less than the interferon gamma receptor activating activity of the interferon gamma polypeptide produced by cleavage of the protease-cleavable linker, and, upon cleavage of both linkers, the in vivo half-life of the interferon gamma polypeptide is substantially similar to the in vivo half-life of naturally occurring interferon gamma. (Item 34) 34. The fusion polypeptide according to any one of items 31 to 33, wherein the protease-cleavable linker polypeptide comprises a sequence capable of being cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloproteases (MMPs), ADAM, FAP, cathepsin L, plasminogen activators, cathepsins, caspases, tryptases, and tumor cell surface proteases. (Item 35) 35. The fusion polypeptide of claim 34, 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 36) 32. The fusion polypeptide of claim 31, wherein the interferon is interferon alpha or interferon gamma. (Item 37) formula [A]-[L1]-[D]-[L2]-[A]-[L2]-[D] [In the formula, A is an interferon polypeptide, L1 and L2 are each independently a protease-cleavable polypeptide linker; D is an interferon blocking moiety that can optionally extend serum half-life; 32. The fusion polypeptide of claim 31, wherein L1 is a substrate for a first protease and L2 is a substrate for a second protease. (Item 38) 38. The fusion polypeptide according to any one of items 31 to 37, wherein the interferon blocking moiety is also a half-life extending element. (Item 39) 38. The fusion polypeptide according to any one of items 31 to 37, wherein the interferon-blocking moiety sterically blocks the agonist activity of the cytokine. (Item 40) 41. The fusion polypeptide of claim 38 or 39, wherein the interferon blocking moiety is human serum albumin or an antigen-binding polypeptide that binds to human serum albumin. 41. The fusion polypeptide according to any one of items 31 to 40, wherein the interferon is freely dissociated from the interferon-blocking moiety after the protease-cleavable polypeptide linker is cleaved by a protease. (Item 42) 42. The fusion polypeptide according to any of items 31 to 41, wherein the interferon-blocking moiety inhibits the interferon polypeptide from activating its cognate receptor. (Item 43) 43. The fusion polypeptide according to any one of items 31 to 42, wherein the fusion polypeptide binds to the cognate receptor of the interferon polypeptide prior to cleavage by the protease-cleavable linker. (Item 44) 44. The fusion polypeptide according to any one of items 31 to 43, further comprising at least one half-life extension element. (Item 45) 45. The fusion polypeptide of any one of items 31 to 44, wherein the interferon-blocking moiety is non-covalently associated with the interferon polypeptide. (Item 46) 45. The fusion polypeptide according to any one of items 31 to 44, wherein the non-covalent association is pH dependent. (Item 47) 45. The fusion polypeptide of item 44, wherein the half-life extending element sterically inhibits or blocks activation and / or binding of the interferon polypeptide to its cognate receptor. (Item 48) 45. The fusion polypeptide according to item 44, wherein the half-life extending element is human serum albumin, human IgG, or a fragment thereof. (Item 49) 49. The fusion polypeptide according to items 44 to 48, wherein the half-life extension element does not bind to the interferon polypeptide. (Item 50) 32. The fusion polypeptide of claim 31, wherein the interferon-blocking moiety comprises a ligand binding domain or fragment of a cognate receptor for the interferon, a single domain antibody or scFv that binds to the interferon polypeptide, or an antibody or antibody fragment (e.g., a single domain antibody, scFv) that binds to a receptor for the interferon. (Item 51) The fusion polypeptide according to any one of claims 31 to 50, wherein the cognate interferon receptor activation activity is determined using a standard in vitro receptor activation assay and equivalent amounts of the fusion polypeptide and interferon polypeptide on a molar basis. 52. The fusion polypeptide according to any one of items 31 to 51, wherein the interferon is freely dissociated from the interferon-blocking moiety and / or half-life extension element after the protease-cleavable sequence is cleaved by a protease. (Item 53) The fusion polypeptide is a polypeptide comprising at least one of [A], [B], and [L] as follows: a) [A]-[L]-[D], b) [D]-[L]-[A], c)[D]-[L]-[A]-[L]-[D]-[L]-[A], d)[A]-[L]-[D]-[L]-[A]-[L]-[D], e) [D]-[L]-[A]-[L]-[D], f)[D]-[L]-[A]-[L]-[A]-[L]-[D], 53. The fusion polypeptide according to any one of items 31 to 52, comprising the fusion polypeptide in any one of the following orientations: g) [D]-[L]-[A]-[L]-[D]-[L]-[A]-[L]-[D]; and h) [D]-[L]-[A]-[A]-[L]-[D]. (Item 54) 54. The fusion polypeptide according to any one of items 31 to 53, wherein the fusion polypeptide comprises two cytokine peptides. (Item 55) 55. The fusion polypeptide of item 54, wherein the two cytokine polypeptides are two different cytokine peptides. (Item 56) 45. The fusion polypeptide according to item 44, wherein the at least one half-life extension element is one half-life extension element or two half-life extension elements. (Item 57) 57. The fusion polypeptide according to any one of items 31 to 56, wherein comprising one protease-cleavable polypeptide linker is one protease-cleavable linker, two protease-cleavable linkers, three protease-cleavable linkers, four protease-cleavable linkers, five protease-cleavable linkers, six protease-cleavable linkers, or seven protease-cleavable linkers. (Item 58) 58. The fusion polypeptide according to any one of items 31 to 57, further comprising a tumor-specific antigen-binding peptide. (Item 59) 59. The fusion polypeptide of claim 58, wherein the tumor-specific antigen-binding peptide is operably linked to the interferon polypeptide by a non-cleavable linker. (Item 60) 59. The fusion polypeptide of claim 58, wherein the tumor-specific antigen-binding peptide is operably linked to the interferon polypeptide by a cleavable linker. (Item 61) A nucleic acid encoding the fusion polypeptide according to any one of items 1 to 60. (Item 62) 62. A vector comprising the nucleic acid according to item 61. (Item 63) A host cell comprising the vector of item 62 or the nucleic acid of item 61. (Item 64) 64. A method for producing a pharmaceutical composition, comprising culturing the host cell according to item 63 under conditions suitable for the expression and harvesting of a desired polypeptide. (Item 65) A pharmaceutical composition comprising i) the fusion polypeptide according to any one of items 1 to 60, and ii) a pharmacologically acceptable additive. (Item 66) 61. A method for treating a tumor, comprising administering to a subject in need thereof an effective amount of the fusion polypeptide according to any one of items 1 to 60. (Item 67) 61. The fusion polypeptide according to any one of items 1 to 60 for use as a medicament. (Item 68) 61. The fusion polypeptide according to any one of items 1 to 60 for use in treating a tumor in a subject in need thereof. (Item 69) 61. 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 60.

Claims

[Claim 1] The invention as depicted in the drawings.

Images