Activatable interleukin-2 polypeptides and use methods thereof

Abstract

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

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 671,225, filed May 14, 2018; U.S. Provisional Application No. 62 / 756,504, filed Nov. 6, 2018; and U.S. Provisional Application No. 62 / 756,507, filed Nov. 6, 2018. The entire teachings of the above applications are hereby incorporated by reference into this specification.

[0002] Sequence Listing This application includes a sequence listing that has been electronically submitted in ASCII format, the entire contents of which are hereby incorporated by reference into this specification. The ASCII copy, created on May 14, 2019, is named 105365-0021_SL.txt and is 408,319 bytes in size.

Background Art

[0003] The development of mature immune lymphoid cells from precursors with low commitment, their subsequent antigen-driven immune responses, and the suppression of these unwanted autoreactive responses are highly dependent on and regulated by cytokines (such as interleukin-2 [IL-2], IL-4, IL-7, IL-9, IL-15, and IL-21, etc.) that utilize receptors of the common γ-chain (γc) family (Rochman et al., 2009) as well as family members such as Il-12, Il-18, and Il-23. IL-2 is essential for the generation of Treg cells in the thymus and critically regulates several important aspects of mature peripheral Tregs and some conventional T cells activated by antigens. IL-2 has been widely studied in part because of its potent T cell proliferative factor activity in vitro, and this activity provides a powerful means to directly boost immunity in patients such as those with cancer and AIDS-HIV, or targets to antagonize unwanted responses such as transplant rejection and autoimmune diseases. In vitro studies using IL-2 have provided strong theoretical basis for these studies, but the in vivo function of IL-2 is clearly much more complex, as first exemplified in IL-2-deficient mice, where a rapid and lethal autoimmune syndrome rather than immune deficiency was observed (Sadlack et al., 1993, 1995). Subsequently, similar observations were 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 activities of some cytokines are well known and described, and some cytokines are already used therapeutically in humans. Among cytokines such as interleukin-2 (IL-2), positive antitumor activities have been shown in patients with various types of tumors, such as renal metastatic carcinomas, hairy cell leukemia, Kaposi's sarcoma, melanoma, multiple myeloma, etc. Other cytokines such as IFNβ, tumor necrosis factor (TNF)α, TNFβ, IL-1, IL-4, IL-6, IL-12, IL-15 and CSF have shown specific antitumor activities against some types of tumors, and thus they are subjects for further research.

Summary of the Invention

Means for Solving the Problems

[0005] This specification provides 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, etc.

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

[0007] In one aspect, there is provided a fusion polypeptide comprising an IL-2 polypeptide, or a functional fragment or mutant protein thereof, and a blocking moiety, such as a steric blocking domain and the like. The blocking moiety is fused to the IL-2 polypeptide either directly or via a linker, and can be separated from the cytokine polypeptide by cleavage of the fusion polypeptide (e.g., protease-mediated cleavage) at the fusion site or within or near the linker or the blocking moiety. For example, when the cytokine polypeptide is fused to the blocking moiety via a linker containing a protease cleavage site, the cytokine polypeptide can be released from the blocking moiety upon cleavage of the linker by protease mediation and can bind to its receptor. The linker is designed to be cleaved at a site of desired cytokine activity, such as within the tumor microenvironment, etc., 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 extension factor. In some embodiments, the fusion polypeptide further comprises a separate serum half-life extension factor. In some embodiments, the fusion polypeptide further comprises a targeting domain. In various embodiments, the serum half-life extension factor is optionally a branched or multi-armed polyethylene glycol (PEG), a full-length human serum albumin (HSA) or a fragment maintaining binding to FcRn, an Fc fragment, or a water-soluble polypeptide such as a nanobody that binds directly to FcRn or binds to human serum albumin.

[0009] In addition to the serum half-life extending factor, the pharmaceutical compositions described herein preferably include one or more target-directed domains that bind to one or more target antigens or one or more regions on a single target antigen. As used herein, the polypeptide constructs of the invention are contemplated to be cleaved, for example, at protease cleavage sites within a disease-specific microenvironment of a subject or in the blood, and the target-directed domain(s) are to bind to a 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. In some embodiments, the target antigen is on a tumor cell, a virus-infected cell, a bacterium-infected cell, a damaged erythrocyte, an arterial plaque cell, or a fibrotic tissue cell.

[0011] The target antigen may optionally be expressed on the surface of an affected cell or tissue, such as a tumor or cancer cell. Examples of tumor target antigens include, but are not limited to, fibroblast activation protein alpha (FAPa), trophoblast glycoprotein (5T4), tumor-associated calcium signal transducer 2 (Trop2), fibronectin EDB (EDB-FN), fibronectin EIIIB domain, CGS-2, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins that contain two antigen-binding domains that bind to two different target antigens known to be expressed on an affected cell or tissue. 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 target-directed polypeptide independently comprises an scFv, a VH domain, a VL domain, a non-Ig domain, or a ligand that specifically binds to a target antigen. In some embodiments, the target-directed polypeptide specifically binds to a cell surface molecule. In some embodiments, the target-directed polypeptide specifically binds to a tumor antigen. In some embodiments, the target-directed polypeptide specifically and independently binds to a tumor antigen selected from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide specifically and independently binds to two different antigens, and at least one of the antigens is a tumor antigen selected from EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and FOLR1. In some embodiments, the target-directed polypeptide functions as a holding domain and is bound to a cytokine via a non-cleavable linker.

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

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

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

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

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

[0018] The present disclosure also relates to the use of conditionally active proteins, nucleic acids encoding conditionally active proteins, vectors or host cells containing such nucleic acids, for the manufacture of a medicament for treating diseases such as proliferative disorders, neoplastic disorders, inflammatory disorders, immune disorders, autoimmune disorders, infectious diseases, viral diseases, allergic reactions, parasitic reactions, graft-versus-host disease or host-versus-graft disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

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Mode for Carrying Out the Invention

[0020] Disclosed herein are methods and compositions for engineering and using constructs containing inducible cytokines. Cytokines are powerful immune agonists, and as such, cytokines are becoming regarded as promising therapeutic agents in oncology. However, cytokines have proven to have a very narrow therapeutic window. Cytokines are thought to have a short half-life in serum and to be extremely potent. As a result, therapeutic administration of cytokines causes undesirable systemic effects and toxicities. These are exacerbated by the need to administer large amounts of cytokine to achieve desired cytokine levels at the site where cytokine action is intended (e.g., a tumor). Unfortunately, due to the biology of cytokines and the inability to effectively target and control their activities, the clinical benefits expected in the treatment of tumors have not been achieved with cytokines.

[0021] This specification discloses a fusion protein that overcomes the problems of toxicity and short half-life that severely restricted the clinical use of cytokines in oncology. The fusion protein contains a cytokine polypeptide having receptor agonist activity. However, in the context of the fusion protein, the cytokine receptor agonist activity is attenuated and the circulating blood half-life is extended. The fusion protein contains protease cleavage sites that are cleaved by proteases associated with the desired site of cytokine activity (e.g., a tumor), typically concentrated or selectively present at the desired site of activity. Thus, the fusion protein preferentially (or selectively) and efficiently cleaves at the desired site of activity 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, releases a form of cytokine from the fusion protein that is much more active as a cytokine receptor agonist (typically at least about 100-fold more active than the fusion protein). The form of cytokine released upon cleavage of the fusion protein typically has a short half-life and is often substantially similar to the half-life of the 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 because the active cytokine is directed to the tumor microenvironment, toxicity is dramatically reduced or eliminated. The fusion proteins described herein enable, for the first time, the administration of a therapeutically effective amount of cytokine to treat tumors that substantially restricts cytokine activity to the tumor microenvironment and dramatically reduces or eliminates the undesirable systemic effects and toxicity of the cytokine.

[0022] Unless otherwise defined, all technical terms, notations, and other scientific terms used in this specification are intended to have meanings commonly understood by those skilled in the art to which the present invention pertains. In some cases, terms with commonly understood meanings are defined in this specification for clarity and / or ready reference, and including such definitions in this specification should not be construed as necessarily representing a departure from what is generally understood in the art. The techniques and procedures described or referenced in this specification are generally well understood and commonly used by those skilled in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Procedures involving the use of commercially available kits and reagents are generally, unless otherwise specified, carried out according to the protocols and conditions defined by the manufacturer.

[0023] "Cytokine" is a well-known technical term that refers to any of a group of immunomodulatory proteins (such as interleukin or interferon) secreted particularly by cells of the immune system and that are regulators of the immune system. Examples of 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; interleukins such as 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-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), and fragments of polypeptides that activate cognate receptors of cytokines (i.e., functional fragments of the foregoing), but are not limited thereto. "Chemokine" is a technical term 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 serum half-life, but retaining receptor agonist activity, typically have a short serum half-life. As used herein, "cytokine with a short half-life" refers to a cytokine that has a substantially short time for circulating in the serum of a subject, e.g., 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 subject's body, and polypeptides having a modified amino acid sequence intended to extend the serum half-life, but retaining receptor agonist activity. Typically, short half-life cytokine polypeptides, such as IL-2 polypeptides, have a serum half-life equivalent to that of naturally occurring IL-2, e.g., within 5-fold, 4-fold, 3-fold, or 2-fold of naturally occurring IL-2. In the latter case, it is not intended to include the addition of heterologous protein domains, e.g., authentic half-life extension elements, such as serum albumin, etc.

[0025] "Sortase" is a peptide transferase that modifies proteins by recognizing and cleaving the carboxyl-terminal localization signal embedded in or attached to the target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 125) (X is any standard amino acid) between the Thr residue and the Gly residue on the target protein, with the Thr residue transiently binding to the Cys residue that 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, displacing Sortase A and linking the two molecules.

[0026] As used herein, the term "steric hindrance substance" 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), etc., via other moieties such as a linker, etc., but does not covalently bind to that cytokine polypeptide otherwise. A steric hindrance substance can bind non-covalently to a cytokine polypeptide, for example, via electrostatic interaction, hydrophobic interaction, ionic bond or hydrogen bond. A steric hindrance substance typically inhibits or blocks the activity of the cytokine moiety by virtue of its proximity to the cytokine moiety and its comparable size. A steric hindrance substance may also block by recruiting large protein binding partners. An example of this is an antibody that binds to serum albumin, and the antibody itself may or may not be large enough to alone block activation or binding, but the recruitment of albumin enables sufficient steric hindrance.

[0027] As used and described herein, a "half-life extension element" is a part of a chimeric polypeptide that extends the serum half-life and improves the pK, for example, by modifying its size (e.g., to exceed the renal filtration cut-off value), shape, hydrodynamic radius, charge, or by modifying parameters such as 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 an additional element from the fusion protein.

[0029] As used herein, a "plasmid" or "viral vector" is a substance that transports the disclosed nucleic acid into a cell in an undegraded state and contains a promoter that results in the expression of a nucleic acid molecule and / or polypeptide in the target cell.

[0030] As used herein, the terms "peptide", "polypeptide", or "protein" are used broadly to mean two or more amino acids linked by peptide bonds. Proteins, peptides, and polypeptides 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 constituting a molecule, and that the peptides of the invention can contain from a few to several more amino acid residues.

[0031] As used throughout, "subject" can be a vertebrate, more specifically, a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. Such terms do not imply a particular age or sex. Thus, both adult and neonatal subjects of either sex are intended to be included.

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

[0033] As used herein, the terms "treat", "treating", or "treatment" refer to a method of affecting a disease or condition or reducing the symptoms of that disease or condition. Thus, in the disclosed methods, treatment can refer to at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially complete reduction in the severity of an established disease or condition or the symptoms of that disease or condition. For example, a method for treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction rate between 10% and 100% compared to the level in nature or the control. It is understood that treatment does not necessarily refer to the cure or complete elimination of the disease, condition, or the symptoms of that disease or condition.

[0034] As used herein, the terms "prevent", "preventing", and "prevention" with respect to a disease or disorder refer to an act, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, which is performed before or substantially simultaneously with the onset of one or more symptoms of the disease or disorder in a subject, and which inhibits or delays the onset or worsening of one or more symptoms of that disease or disorder.

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

[0036] A "attenuated cytokine receptor agonist" is a cytokine receptor agonist having a reduced receptor agonist activity as compared to the naturally occurring agonist of the cytokine receptor. The attenuated cytokine agonist may have an agonist activity that 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, at least about 1000-fold or lower as compared to the naturally occurring agonist of the receptor. When a fusion protein containing a cytokine polypeptide described herein is described as having "attenuated" or "attenuated activity", it means that the fusion protein is an attenuated cytokine receptor agonist.

[0037] An "intact fusion protein" is, for example, a fusion protein that has no domain removed by cleavage by a protease or the like. The domain may be removable by cleavage by a protease or other enzymatic activity, but this has not occurred when the fusion protein is "intact".

[0038] As used herein, "moiety" refers to a portion of a molecule that has a distinct function within the molecule and whose function can be carried out by that portion in the context of another molecule. The moiety may be a chemical substance having a specific function or a portion of a biological molecule having 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. This may be a protein domain such as serum albumin. Blocking may be achieved by a steric blocker or a specific blocker. A steric blocker blocks by size and position rather than based on a specific binding, and an example is serum albumin. A specific blocker blocks by a specific interaction with the moiety to be blocked. The specific blocker needs to be tailored to a specific cytokine or active domain, and the steric blocker can be used regardless of the payload as long as it is of sufficient size.

[0039] Generally, 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 its in vitro cytotoxic effects on various tumor lines, but it was later proven to have strong pro-inflammatory activity and, when in a state of overproduction, can dangerously affect the human body. Since systemic toxicity is a fundamental problem associated with the use of pharmacologically active amounts of cytokines in humans, new derivatives and therapeutic strategies aimed at reducing their toxic effects while maintaining the therapeutic efficacy of this class of biological effectors are currently under evaluation.

[0040] IL-2 exerts both stimulatory and regulatory functions in the immune system and is central to immune homeostasis, along with other members of the common γ-chain (γc) cytokine family. 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) chain, the IL-2Rβ (CD122) chain, and the IL-2Rγ (γc, CD132) chain or a dimeric βγ IL-2R (1, 3). Any IL-2R variant can transmit a signal upon IL-2 binding. However, the trimeric αβγ IL-2R has an affinity for IL-2 that is approximately 10 - 100 times higher than that of the dimeric βγ IL-2R (3), and it is suggested that CD25 confers high-affinity binding of IL-2 to its receptor but is not essential for signal transduction. The trimeric IL-2R is found on activated T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg), which are sensitive to IL-2 in vitro and in vivo. Conversely, antigen-experienced (memory) CD8+, CD44high memory phenotype (MP) CD8+, and natural killer (NK) cells are provided with high levels of the dimeric βγ IL-2R, and these cells also respond vigorously to IL-2 in vitro and in vivo.

[0041] The expression of high-affinity IL-2R is important for T cells to respond to low concentrations of IL-2 that are transiently available in vivo. The expression of IL-2Rα is not seen in naive T cells 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 not as strictly regulated and is constitutively expressed by all lymphoid cells. Once high-affinity IL-2R is induced by antigen, IL-2R signaling increases the expression of IL-2Rα, in part, via Stat5-dependent regulation of Il2ra transcription (Kim et al., 2001). This process represents a mechanism to maintain the expression of high-affinity IL-2R and sustain IL-2 signaling while the IL-2 source remains.

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

[0043] In other words, the IL-2Rα-IL-2Rβ-IL-2 ternary complex then recruits γc via weak interaction with IL-2 and stronger interaction with IL-2Rβ to generate a stable quaternary high-affinity IL-2R (Kd 10-11 M, 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, but IL-2Rα is recycled to the cell surface (Hemar et al., 1995, Yu and Malek, 2001). Thus, for those functional activities that require sustained IL-2R signal transduction, a continuous source of IL-2 is required to associate with IL-2Rα to form additional IL-2-IL-2R signal transduction complexes.

[0044] Regulatory T cells actively suppress the activation of the immune system and prevent pathological autoreactive diseases and the resulting autoimmune diseases. The development of drugs and methods for selectively activating regulatory T cells for the treatment of autoimmune diseases has been a major subject of intensive research and, until the development of the present invention, which can selectively deliver active interleukins to the site of inflammation, has been mostly unsuccessful. 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 the immune response to foreign antigens. Multiple 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.

[0045] As a result, there is great interest in the development of therapies that enhance the number and / or function of Treg cells. One of the autoimmune disease treatment methods being investigated 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 been shown to be promising in the treatment of animal models of disease and in some early-stage human clinical trials, but requires personalized medicine using the patient's own T cells, is invasive, and is technically complex. Another method is treatment with low-dose interleukin-2 (IL-2). Treg cells are characteristically expressed at 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 the proliferation of Treg cells has been shown to be IL-2 dependent (Malek, T.R., et al., 2010, Immunity, 33:153-65).

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

[0047] In clinical trials of low-dose IL-2 therapy for chronic GVHD (Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and patients with HCV-related autoimmune vasculitis (Saadoun, D., et al., 2011, N Engl J Med., 365:2067-77), an increase in Treg levels and signs of clinical efficacy have been demonstrated. New clinical trials are underway to investigate the efficacy of IL-2 in multiple other autoimmune and inflammatory diseases. The theoretical basis for using so-called low-dose IL-2 is to utilize the high IL-2 affinity of the trimeric IL-2 receptor that is constitutively expressed on Tregs, while keeping other T cells that do not express the high-affinity receptor in an inactivated state. Aldesleukin, a recombinant form of IL-2 used in these trials (sold as Proleukin® by Prometheus Laboratories, San Diego, CA), is associated with high toxicity. Aldesleukin is approved for the treatment of metastatic melanoma and metastatic renal cell carcinoma, but its side effects are very severe, so its use is recommended only in hospitals where intensive treatment can be provided (web address: www.proleukin.com / assets / pdf / proleukin.pdf).

[0048] Treg cells respond to lower concentrations of IL-2 than many other immune cell types by virtue of their expression of IL2R alpha. Therefore, in clinical trials of IL-2 for autoimmune diseases, lower doses of IL-2 have been used to target Treg cells (Klatzmann D, 2015 Nat Rev Immunol.15:283-94). However, even these low doses have raised safety and tolerability issues, and the treatments used employed either daily subcutaneous injections or a chronic or intermittent 5-day treatment course. Therefore, there is a need for a treatment for autoimmune diseases that enhances the number and function of Treg cells, targets Treg cells more specifically than IL-2, is safer and more tolerable, and has a lower dosing frequency.

[0049] One approach that has been proposed to improve the therapeutic index of IL-2-based therapies for autoimmune diseases is to use a mutant form of IL-2 that is selective for Treg cells compared to other immune cells. The IL-2 receptor is expressed on a diverse range of immune cell types, such as T cells, NK cells, eosinophils, and monocytes, and this broad expression pattern may contribute to its pleiotropic effects on the immune system and high systemic toxicity. In particular, activated effector T cells, like lung epithelial cells, express IL2Rαβγ. However, the activation of effector T cells is directly opposed to the goal of suppressing and controlling the immune response, and the 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 the accumulation of intravascular fluid in organs such as the lungs and liver, leading to subsequent pulmonary edema and hepatocyte damage. There is no treatment for VLS other than discontinuation of IL-2. To avoid VLS, low-dose IL-2 regimens have been tested in patients, but the trade-off is that the treatment outcomes are not optimal.

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

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

[0052] The direct use of IL-2 as an agonist that binds to IL-2R and therapeutically regulates the immune response has problems due to its well-documented therapeutic risks, such as the short half-life of its serum and high toxicity. These risks have also limited the development of treatment methods and the use of other cytokines. New forms of cytokines are needed to reduce these risks. This specification discloses compositions and methods that include IL-2 and IL-15 and other cytokines, functional fragments and mutant proteins of cytokines, and conditionally active cytokines designed to address these risks and provide the required immunomodulatory treatment methods.

[0053] The present invention is designed to address the drawbacks of direct IL-2 therapy and therapies using other cytokines, and uses, for example, cytokine blocking moieties such as steric blocking polypeptides, serum half-life extended polypeptides, targeting polypeptides, linker polypeptides such as protease-cleavable linkers, and combinations thereof. Cytokines are extremely potent when administered to a patient, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons (IFNs such as IFN alpha, IFN beta, and IFN gamma), tumor necrosis factors (e.g., TNF alpha, lymphotoxin), transforming growth factors (e.g., TGF beta1, TGF beta2, TGF beta3), chemokines (C-X-C motif chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte macrophage colony-stimulating factor (GM-CS). As used herein, "chemokine" means a family of small cytokines capable of inducing chemotaxis directed towards the vicinity of responsive cells. Cytokines can provide powerful therapies but are associated with undesirable effects that are clinically difficult to manage, thereby limiting their clinical use. The present disclosure relates to new forms of cytokines that can be used in patients in which the undesirable effects are reduced or eliminated. In particular, the present disclosure relates to pharmaceutical compositions comprising chimeric polypeptides (fusion proteins), nucleic acids encoding the fusion proteins, and the aforementioned pharmaceutical formulations containing 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 and often have the same or higher potency than the corresponding naturally occurring cytokines. As described herein, this is typically achieved by using a cytokine blocking moiety that blocks or inhibits the receptor activation function of the cytokine, its active fragment, or mutant protein, under general conditions rather than under selected conditions such as those present at the desired site of cytokine activity (e.g., the site of inflammation or tumor).

[0054] Chimeric polypeptides and nucleic acids encoding chimeric polypeptides can be produced using any suitable method. For example, nucleic acids encoding chimeric polypeptides can be produced using recombinant DNA technology, synthetic chemistry, or a combination of these techniques, and can be expressed in a suitable expression system such as CHO cells. Chimeric polypeptides can likewise be produced, for example, by expression of a suitable nucleic acid using synthetic or semi-synthetic chemical techniques and the like. In some embodiments, the blocking moiety can be attached to the cytokine polypeptide via sortase-mediated linkage. “Sortase” is a peptide transferase that modifies proteins by recognizing and cleaving a carboxyl-terminal localization signal that is embedded within or attached to the target protein or peptide. Sortase A catalyzes the cleavage of the LPXTG motif (SEQ ID NO: 125) (X is any standard amino acid) between a Thr residue and a Gly residue on the target protein, with the Thr residue transiently binding to a Cys residue that 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 having an N-terminal nucleophile, typically an oligoglycine motif, attacks the intermediate, displacing sortase A and linking the two molecules.

[0055] To form the cytokine blocking partial fusion protein, the cytokine polypeptide is first tagged with a polyglycine sequence at the N-terminus or alternatively tagged with an LPXTG motif (SEQ ID NO: 125) at the C-terminus. A peptide is conjugated to each of the blocking portion or other elements, functioning as an acceptor site for the tagged polypeptide. In the case of conjugation to a domain that holds an LPXTG acceptor peptide (SEQ ID NO: 125) conjugated through its own N-terminus, the polypeptide is tagged with an N-terminal polyglycine stretch. In the case of conjugation to a domain that holds a polyglycine peptide conjugated through its own C-terminus, the polypeptide is tagged with an LPXTG sortase recognition sequence (SEQ ID NO: 125) at its C-terminus. When the sortase recognizes the polyglycine sequence and the LPXTG (SEQ ID NO: 125) sequence, it forms a peptide bond between the polymer peptide and the tagged polypeptide. The sortase reaction cleaves the glycine residue as an intermediate, which occurs at room temperature.

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

[0057] Any suitable linker can be used. For example, the linker can be glycine-glycine, a sortase recognition motif, or a combination of a sortase recognition motif and a peptide sequence (Gly4Ser) n (SEQ ID NO: 126) or (Gly3Ser) n(SEQ ID NO: 127) (where n can be 1, 2, 3, 4, or 5) can be included. Typically, the sortase recognition motif includes the peptide sequence LPXTG (SEQ ID NO: 125), where X is any amino acid. In some embodiments, the covalent bond is between a reactive lysine residue bound to the C-terminus of the cytokine polypeptide and a reactive aspartic acid bound to the N-terminus of the blocking substance or other domain. In other embodiments, the covalent bond is between a reactive aspartic acid residue bound to the N-terminus of the cytokine polypeptide and a reactive lysine residue bound to the C-terminus of the blocking substance or other domain.

[0058] Thus, as detailed herein, the cytokine blocking moiety used (e.g., the IL-2 blocking moiety) can be a steric blocking substance. As used herein, a "steric blocking substance" refers to a polypeptide or polypeptide moiety that can be covalently bound directly or indirectly to a cytokine polypeptide, e.g., in the form of a chimeric polypeptide (fusion protein), etc., via another moiety such as a linker, but that does not covalently bind to that cytokine polypeptide otherwise. A steric blocking substance can bind non-covalently to a cytokine polypeptide, e.g., via electrostatic, hydrophobic, ionic, or hydrogen bonds. A steric blocking substance typically inhibits or blocks the activity of the cytokine moiety by virtue of its proximity to the cytokine moiety and its comparable size. The steric hindrance of the cytokine moiety can be removed by spatially separating the steric blocking substance from the cytokine moiety, e.g., by enzymatically cleaving a fusion protein containing the steric blocking substance and the cytokine polypeptide at a site between the steric blocking substance and the cytokine polypeptide.

[0059] As will be described in more detail herein, the blocking function may be in combination with additional functional components in the pharmaceutical composition, such as a targeting domain, a serum half-life extension factor, and a protease-cleavable linker polypeptide, etc., or may be due to their presence. For example, a serum half-life extension polypeptide may also be a steric blocker.

[0060] Various factors ensure the delivery and activity of IL-2 preferentially to the site of desired IL-2 activity and severely limit the systemic exposure to interleukin through a blocking and / or targeting strategy preferentially linked to a serum half-life extension strategy. In this serum half-life extension strategy, the blocking form of interleukin circulates for a long time (preferably 1 to 2 weeks or more), while the activated form has the typical serum half-life of interleukin.

[0061] When compared to the serum half-life extended type, the serum half-life of intravenously administered IL-2 is only about 10 minutes because it is distributed in the extracellular space of the whole body, which is as large as about 15 L in an average-sized adult. Thereafter, IL-2 is metabolized by the kidneys and the half-life is about 2.5 hours. (Smith, K. “Interleukin 2 immunotherapy”. Therapeutic Immunology 240 (2001)). In other measurements, the plasma half-life of IL-2 is very short, 85 minutes for intravenous administration and 3.3 hours for subcutaneous administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol. 46:5 - 10). In some embodiments of the present invention, the half-life extension element is linked to the interleukin via a linker and is cleaved at the site of action (e.g., by an inflammation-specific or tumor-specific protease) to release the full activity of the interleukin at the desired site and also to separate it from the uncleaved half-life extension. In such embodiments, the fully active free interleukin has very different pharmacokinetic (pK) properties and the half-life is hours rather than weeks. Furthermore, the exposure to the active cytokine is limited to the site of the desired cytokine activity (e.g., the site of inflammation or tumor), and the systemic exposure to the active cytokine, as well as the associated toxicity and side effects, are reduced.

[0062] Other cytokines contemplated in the present invention have pharmacology similar to that of 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 drawbacks of directly using these agents, provides chimeric polypeptides that can have a longer half-life and / or be directed to the site of the desired activity (e.g., the site of inflammation or tumor).

[0063] If desired, IL-2 can be engineered to generally bind to the IL-2R complex or specifically bind to one of the three IL-2R subunits with an affinity different from that of the corresponding wild-type IL-2, for example, 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 IL-2 receptor, as compared to wild-type IL-2, can have an amino acid sequence containing 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, 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, 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 mutant proteins of other cytokines, such as interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23), interferons (IFNs such as IFN alpha, IFN beta, and IFN gamma), tumor necrosis factors (e.g., TNF alpha, 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, mutant proteins having a desired binding affinity for homologous receptors can be prepared.

[0064] As described above, any of the mutant IL-2 polypeptides disclosed herein can include the recited sequences, and they can be limited to the recited sequences or, in other cases, can be identical to SEQ ID NO:1. Further, any of the mutant IL-2 polypeptides disclosed herein can optionally include a substitution of another residue (e.g., serine) for the cysteine residue at position 125, and / or can optionally include a deletion of the alanine residue at position 1 of SEQ ID NO:1.

[0065] Another way to improve the therapeutic index of IL-2-based therapies is to optimize the pharmacokinetics of the molecule to maximize Treg cell activation. Initial studies of IL-2 action demonstrated that IL-2 stimulation of human T cell proliferation in vitro required a minimum 5- to 6-hour exposure to effective concentrations of IL-2 (Cantrell, D.A., et.al., 1984, Science, 224:1312-1316). When administered to human patients, IL-2 has a very short plasma half-life of 85 minutes following intravenous administration and 3.3 hours following subcutaneous administration (Kirchner, G.I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Because of its short half-life, maintaining circulating IL-2 above the levels required to stimulate T cell proliferation for the period necessary requires either high doses that result in IL-2 peak levels well above the EC50 of Treg cells or frequent dosing. These high IL-2 peak levels can activate the IL2Rβγ receptor and can result in other unintended or harmful effects, such as VLS as described above. Multifunctional proteins with a longer circulating plasma half-life than IL-2, such as IL-2 analogs or IL-2 bound to a domain that enables binding to the FcRn receptor, can achieve the desired drug concentration at lower doses and lower peak levels than IL-2 for a specified period. Thus, such IL-2 analogs require only lower doses or less frequent administration than IL-2 to effectively stimulate Treg cells. Less frequent subcutaneous administration of IL-2 drugs is also more tolerable for patients. Therapies with these characteristics will clinically lead to improved pharmacological efficacy, reduced toxicity, and improved patient compliance for the therapy. Alternatively, IL-2 or a mutant protein of IL-2 (herein, “IL-2*”) can be selectively targeted to the intended site of action (e.g., the site of inflammation). This targeting can be achieved by one of several strategies, such as the addition of a domain to the administered agent that includes a blocking substance for the cleaved IL-2 (or mutant protein), or by targeting the domain, or by a combination of the two.

[0066] In some embodiments, the IL-2* partial agonist can be engineered to bind with higher or lower affinity depending on the desired target. For example, IL-2* can be engineered to bind with enhanced affinity to one of the receptor subunits and not to the other subunits. These types of partial agonists, unlike full agonists or full antagonists, provide the ability to tune signaling properties to an amplitude that induces desired functional properties without crossing a threshold of undesired properties. Given the specific activity of partial agonists, the repertoire of IL-2 variants can be engineered to exhibit a more finely graded characteristic signaling activity ranging from almost full- to partial-agonist activity to full-antagonist activity.

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

[0068] The blocking moieties described in detail below can also be used to promote binding or activation to one or more receptors. In one embodiment, the blocking moiety is added such that binding or activation of IL-2Rβγ is blocked but binding or activation of IL-2Rα is not altered. In another embodiment, the blocking moiety is added such that binding or activation of IL-2Rα is decreased. In another embodiment, the blocking moiety is added such that binding and / or activation of all three receptors is inhibited. This blockade can be alleviated by removal of the blocking moiety in a particular context, for example, by proteolytic cleavage of a linker that couples one or more blocking moieties to the cytokine.

[0069] Similar methods can be applied to improve other cytokines for use, for example, in cancer treatment, particularly as immunostimulants. For example, in this aspect, the pharmacokinetics and / or pharmacodynamics of cytokines (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFN alpha, IFN beta, and IFN gamma, TNF alpha, lymphotoxin, TGF beta1, TGF beta2, TGF beta3 GM-CSF, CXCL10, CCL19, CCL20, and CCL21 etc.) can be adjusted to maximally activate effector cells (e.g., effector T cells, NK cells) and / or cells that promote a cytotoxic immune response at the site of desired activity, such as within a tumor, preferably not systemically, for example, by inducing maturation of dendritic cells.

[0070] Accordingly, provided herein is a pharmaceutical composition comprising at least one cytokine polypeptide, such as interleukin (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferon (IFN such as IFN alpha, IFN beta, and IFN gamma), tumor necrosis factor (e.g., TNF alpha, lymphotoxin), transforming growth factor (e.g., TGF beta 1, TGF beta 2, TGF beta 3), chemokine (e.g., CXCL10, CCL19, CCL20, CCL21), and granulocyte macrophage colony stimulating factor (GM-CS), etc., or a functional fragment or mutant protein of any of the foregoing. The polypeptide typically further comprises at least one linker amino acid sequence, and the amino acid sequence can be cleaved by an endogenous protease in certain embodiments. In one embodiment, the linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130), or SGESPAYYTA (SEQ ID NO: 131). In other embodiments, the chimeric polypeptide further contains a blocking portion capable of blocking the activity of the interleukin polypeptide, such as a steric blocking polypeptide portion, etc. The blocking portion 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 portion, such as a steric blocking polypeptide portion, and the blocking portion blocks the activity of the cytokine polypeptide on the cytokine receptor, and in certain embodiments, the blocking portion comprises a protease-cleavable domain. In some embodiments, the blocking and reduction of cytokine activity is readily achieved by attaching an additional domain to the N-terminus or C-terminus of the interleukin domain using a very short linker. In such embodiments, it is expected that the blocking is reduced by the short linker that tethers the blocking portion or the blocking substance to the interleukin being digested by a protease.Once the domain is excised or released, it becomes impossible to achieve blockade of cytokine activity.

[0071] A pharmaceutical composition, e.g., a chimeric polypeptide, can comprise two or more cytokines, which can be either 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 chemotactic agent, e.g., CXCL10, and the second cytokine is an immune cell activator.

[0072] Preferably, the cytokine polypeptides (including functional fragments) contained in the fusion proteins disclosed herein are not mutated or engineered to alter the properties of the 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 allowed, for example, to facilitate cloning and to achieve the desired expression level.

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

[0074] Blocking part The blocking moiety can be any moiety that inhibits the ability of a cytokine to bind to and / or activate its receptor. The blocking moiety can inhibit the ability of a cytokine to bind to and / or activate its receptor by sterically blocking it and / or by covalently binding to the cytokine. Examples of suitable blocking moieties include full-length or cytokine-binding fragments or mutant proteins of the cytokine's cognate receptor. Antibodies and fragments thereof such as polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (variable domains of heavy-chain variable domains (VH), light-chain variable domains (VL), and camelid nanobodies (VHH), etc.), dAbs, etc. that bind to a cytokine can also be used. Other suitable antigen-binding domains that bind to a cytokine can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of an antibody, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, knotted domain peptides, monobodies, as well as binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of suitable blocking polypeptides include polypeptides that sterically inhibit or block the binding of a cytokine to its cognate receptor. Advantageously, such moieties also function as half-life extension elements. For example, a peptide modified by conjugation to a water-soluble polymer such as PEG can sterically inhibit or prevent the binding of a cytokine to its receptor. Polypeptides or fragments thereof with a long half-life in serum, such as serum albumin (human serum albumin), immunoglobulin Fc, transferrin, etc., as well as fragments and mutant proteins of such polypeptides, can also be used. For example, antibodies and antigen-binding domains that bind to proteins with a long half-life in serum such as HSA, immunoglobulin, or transferrin, or that bind to receptors recycled to the plasma membrane such as FcRn or the transferrin receptor, can also inhibit a cytokine, particularly when bound to their antigen.Examples of such antigen-binding polypeptides include single-chain variable fragments (scFv), single-domain antibodies (variable domains of heavy-chain variable domains (VH), light-chain variable domains (VL), and camelid nanobodies (VHH), etc.), dAbs, etc. Other suitable antigen-binding domains that bind to cytokines can also be used, including non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, such as anticalins, affilins, affibody molecules, affimers, affitins, alphabodies, avimers, DARPins, fynomers, knotted domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4, etc.

[0075] In an exemplary example, when IL-2 is the cytokine in the chimeric polypeptide, the blocking moiety can be the full-length or fragment or mutant protein of the alpha chain of the IL-2 receptor (IL-2Rα) or the beta (IL-2Rβ) or gamma chain (IL-2Rγ) of the IL-2 receptor, an anti-IL-2 single-domain antibody (dAb) or scFv, Fab, an anti-CD25 antibody or a fragment thereof, and an anti-HAS dAb or scFv, etc.

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

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

[0078] 3. The scaffolding that has been engineered is the fusion protein according to embodiment 1 or 2, which comprises sdAb, scFv, Fab, VHH, fibronectin type III domain, immunoglobulin-like scaffolding, DARPin, cystine knot peptide, lipocalin, three-helix bundle scaffolding, protein G-related albumin-binding module, or DNA or RNA aptamer scaffolding.

[0079] 4. The binding moiety is the fusion protein according to any one of embodiments 1 to 2, which can bind to bulk serum proteins.

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

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

[0082] 7. The binding moiety is the fusion protein according to embodiment 5, which can bind to the bulk serum protein.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0097] 22. The fusion protein according to embodiment 21, wherein the protease cleavage site is recognized by a serine protease, a cysteine protease, an aspartic protease, a threonine protease, a glutamic protease, a metalloprotease, a collagenase, or an asparaginyl peptidase.

[0098] 23. The fusion protein according to embodiment 21, wherein the protease cleavage site is recognized by 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, actinidin, 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 metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

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

[0100] 25. The conditionally active binding protein according to embodiment 24, wherein the binding moiety is capable of binding to a half-life extending protein.

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

[0102] 27. The conditionally active binding protein according to embodiment 26, wherein the engineered scaffold comprises an sdAb, an scFv, a Fab, a VHH, a 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.

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

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

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

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

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

[0108] 33. The binding moiety of the conditionally active binding protein according to any one of embodiments 29 to 32 can mask the binding of the cytokine to its target through specific intermolecular interactions between the binding moiety and the cytokine.

[0109] 34. The non-CDR loop of the conditionally active binding protein according to any one of embodiments 29 to 33 provides a binding site specific to the binding of the binding moiety to the cytokine.

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

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

[0112] 37. The cleavable linker of the conditionally active binding protein according to embodiments 24 to 36 includes a cleavage site.

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

[0114] 39. The protease cleavage site is a conditionally active binding protein according to embodiment 38, which is recognized by serine protease, cysteine protease, aspartic protease, threonine protease, glutamic protease, metalloprotease, gelatinase, or asparagine peptidase.

[0115] 40. The protease cleavage site is a conditionally active binding protein according to embodiment 38, which is recognized by 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, actinidin, 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 metalloprotease (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, ADAM10, ADAM17, ADAM12, urokinase-type plasminogen activator (uPA), enterokinase, prostate-specific target (PSA, hK3), interleukin-1β converting enzyme, thrombin, FAP (FAP-α), dipeptidyl peptidase, or dipeptidyl peptidase IV (DPPIV / CD26), type II transmembrane serine protease (TTSP), neutrophil elastase, cathepsin G, proteinase 3, neutrophil serine protease 4, mast cell chymase, mast cell tryptase, dipeptidyl peptidase, and dipeptidyl peptidase IV (DPPIV / CD26).

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

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

[0118] 43. A conditionally active binding protein comprising the binding portion that binds to a cytokine via a non-CDR loop within the binding portion, wherein the binding portion is further linked to a half-life extension domain and comprises a cleavable linker, and the binding protein has an extended half-life prior to its activation by cleavage of the linker, and upon activation, the binding portion and the half-life extension domain are separated from the cytokine, and the binding protein has an unextended half-life in its activated state, the conditionally active binding protein.

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

[0120] In vivo half-life extension element Preferably, the chimeric polypeptide comprises an in vivo half-life extension element. By increasing the in vivo half-life of a therapeutic molecule that is naturally short-lived, a more tolerable and manageable dosing regimen becomes possible without sacrificing efficacy. As used herein, an "in vivo half-life extension element" is, for example, a part of a chimeric polypeptide that increases the in vivo half-life and improves the pK by modifying its size (e.g., to exceed the renal filtration cut-off value), shape, hydrodynamic radius, charge, or by modifying parameters such as absorption, biodistribution, metabolism, and elimination. An exemplary method of improving the pK of a polypeptide is by the expression of an element of the polypeptide chain that binds to a receptor that is recycled to the cell plasma membrane rather than degraded in the lysosome, such as the FcRn receptor and the transferrin receptor on endothelial cells. Three proteins, for example, human IgG, HSA (or fragment), and transferrin, persist in human serum much longer than predicted from their size alone, which is due to their ability to bind to receptors that are recycled rather than degraded in the lysosome. These proteins, or fragments thereof that retain FcRn binding, are routinely conjugated to other polypeptides to extend their serum half-life. In one embodiment, the in vivo half-life extension element is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO: 2) may also be conjugated directly to the pharmaceutical composition or via a short linker. Fragments of HSA may be used. HSA and its fragments can function both as a blocking moiety and as an in vivo half-life extension element. Human IgG and Fc fragments can also perform a similar function.

[0121] The serum half-life extending factor can also be an antigen-binding polypeptide that binds to proteins with a long serum half-life such as serum albumin and transferrin. 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 (variable domains of heavy-chain variable domains (VH), light-chain variable domains (VL), and camelid nanobodies (VHH), etc.), 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, knotted domain peptides, monobodies, as well as binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding portions of receptors, lectins, and peptides that bind to or associate with one or more target antigens.

[0122] Some preferred serum half-life extension elements are polypeptides that include complementarity determining regions (CDRs) and optionally non-CDR loops. Advantageously, such serum half-life extension elements can extend the serum half-life of cytokines and also function as cytokine inhibitors (e.g., via steric hindrance, non-covalent interactions, or combinations thereof) and / or as targeting domains. In some cases, the serum half-life extension element is a domain derived from an immunoglobulin molecule (Ig molecule) or an engineered protein scaffold that mimics the structure and / or binding activity of an antibody. The Ig can be of any class or subclass (IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, etc.). The polypeptide chains of the Ig molecule fold into a series of parallel beta strands linked by loops. In the variable region, three of the loops constitute the "complementarity determining regions" (CDRs) that determine the antigen-binding specificity of the molecule. An IgG molecule includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding fragments thereof. 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 have sequences that are hypervariable and / or are involved in antigen recognition and / or form loops that are typically structurally defined, interspersed with more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 from the amino terminus to the carboxy terminus. In some embodiments of the present disclosure, at least a portion or all of the amino acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR loops" of the binding moiety described herein. As shown in FIG. 5, the variable domain of an immunoglobulin molecule has several beta strands arranged in two sheet-like structures.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 domain form a cluster at one end of the beta barrel. The CDRs are loops that connect the beta strands B-C, C’-C”, and F-G of the immunoglobulin fold, while the lower loops that connect the beta strands AB, CC’, C”-D, and E-F of the immunoglobulin fold, as well as the upper loop that connects the D-E strand of the immunoglobulin fold, are non-CDR loops. In some embodiments of the present disclosure, at least some amino acid residues of the constant 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 AB, CD, EF, and DE loops of the C1 set domains of Ig or Ig-like molecules; the AB, CC’, EF, FG, BC, and EC’ loops of the C2 set domains of Ig or Ig-like molecules; the DE, BD, GF, A(A1A2)B, and EF loops of the I (intermediate) set domains of Ig or Ig-like molecules.

[0123] Within the variable domain, the CDRs are thought to be involved in antigen recognition and binding, and the FR residues are considered to be the scaffolds for the CDRs. However, in certain cases, some of the FR residues play important roles in antigen recognition and binding. Residues in the framework region that affect Ag binding can be divided into two categories. The first are FR residues that contact the antigen, and thus they are part of the binding site, and some of these residues are adjacent and aligned with the CDRs. The other residues are aligned away from the CDRs but are proximal in the 3-D structure of the molecule, for example, loops of the heavy chain. Serum half-life extension domains (e.g., domains that include CDRs) can include at least one non-CDR loop. In some embodiments, the non-CDR loops provide a binding site for binding to cytokines, bulk serum proteins, or other target antigens.

[0124] The serum half-life extension factor contains, in addition to, or alternatively to, a CDR, 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 like albumin, or for a cytokine moiety or other target antigen. To modify the non-CDR loop, it is contemplated that various techniques can be used, such as site-directed mutagenesis, random mutagenesis, insertion of at least one amino acid that is 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. The modification to generate the antigen-binding site may, in some cases, be 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 FIG. 5, i.e., AB, CC’, C”D, EF, and D-E. In some cases, the modification is within the DE loop. In other cases, the modification is in all four loops of AB, CC’, C”-D, and E-F.

[0125] In some examples, the serum half-life extension factor 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 and blocks the cytokine domain. In other examples, the serum half-life extension factor 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 extension factor inhibits the binding of the cytokine to its cognate cytokine receptor, for example, through steric hindrance, through specific intermolecular interactions, or a combination of both.

[0126] In some embodiments, the serum half-life extension factor binds directly to the cytokine non-covalently and inhibits its activity.

[0127] In certain examples, the binding moiety binds to the cytokine via one or more of AB, CC′, C″D, and E-F loops, and binds to a bulk serum protein such as albumin via one or more of BC, C′C″, and FG loops. In certain examples, the binding moiety binds to a bulk serum protein such as albumin via its AB, CC′, C″D, or EF loop, and binds to the cytokine via its BC, C′C″, or FG loop. In certain examples, the binding moiety binds to a bulk serum protein such as albumin via its AB, CC′, C″D, and EF loops, and is bound to the cytokine via its BC, C′C″, and FG loops. In certain examples, the binding moiety binds to a bulk serum protein such as albumin via one or more of AB, CC′, C″D, and E-F loops, and binds to the cytokine via one or more of BC, C′C″, and FG loops.

[0128] The binding moiety is a polypeptide of any kind. 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.

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

[0130] One example of an exemplary conditionally active protein is shown in FIG. 6. In the illustrated example, a non-CDR loop within a serum albumin binding domain (e.g., 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 CDR of the serum albumin binding domain.

[0131] The serum half-life extending element can be any type of binding domain, including but not limited to domains from monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies. In some embodiments, the binding moiety is a single-chain variable fragment (scFv), a single-domain antibody, such as a heavy-chain variable domain (VH), a light-chain variable domain (VL), and a variable domain of a camelid-derived nanobody (VHH), etc. In other embodiments, the binding moiety is a non-Ig binding domain, i.e., an antibody mimetic such as an anticalin, an affilin, an affibody molecule, an affimer, an affitin, an alphabody, an avimer, a DARPin, a fynomer, a knotted domain peptide, and a monobody.

[0132] 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)" have the same meaning and include any non-peptide water-soluble poly(ethylene oxide). The term "PEG" also means a polymer that contains, for the most part, i.e., more than 50%, -OCH2CH2- repeating subunits. For certain forms, PEG can take on any number of different molecular weights, as well as structures or geometries, such as "branched", "linear", "forked", "multifunctional", etc., details of which are described 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-arm type (e.g., forked PEG or PEG attached to a polyol core), dendritic (or star-shaped) structures, with or without one or more degradable linkages in each case. Furthermore, the internal structure of PEG can be organized in any number of different repeating patterns and can also be selected from the group consisting of homopolymers, alternating copolymers, random copolymers, block copolymers, alternating terpolymers, random terpolymers, and block terpolymers. PEG can be conjugated to polypeptides and peptides by any suitable method. Typically, a reactive PEG derivative such as N-hydroxysuccinamidyl ester PEG is reacted with a peptide or polypeptide that contains amino acids with side chains containing amine, sulfhydryl, carboxylic acid, or hydroxyl functional groups such as cysteine, lysine, asparagine, glutamine, theonine, tyrosine, serine, aspartic acid, and glutamic acid. Targeting domain and retention domain

[0133] For certain applications, it may be desirable to maximize the time that the construct is present at its desired site within the body. This can be achieved by including an additional domain in the chimeric polypeptide (fusion protein) that affects its movement within the body. For example, a chimeric nucleic acid can encode a domain that directs the polypeptide to a site within the body, such as a tumor cell or an inflammatory site (this domain is referred to as a "targeting domain"), and / or a domain that retains the polypeptide at a site within the body, such as a tumor cell or an inflammatory site (this domain is referred to as a "retention domain"). In some embodiments, the domain can function as both a targeting domain and a retention domain. In some embodiments, the targeting domain and / or the retention domain is specific for an environment rich in proteases. In some embodiments, the encoded targeting domain and / or retention domain is specific for regulatory T cells (Tregs), for example, targeting the CCR4 receptor or the CD39 receptor. Other suitable targeting domains and / or retention domains include those having cognate ligands that are overexpressed in inflamed tissue, such as the cognate ligand of the IL-1 receptor or the IL-6 receptor. In other embodiments, suitable targeting domains and / or retention domains include those having cognate ligands that are overexpressed in tumor tissue, such as the cognate ligand of Epcam, CEA or mesothelin. In some embodiments, the targeting domain is linked to the interleukin via a linker and is cleaved at the site of action (e.g., by a protease specific for inflammation or cancer) to release the full activity of the interleukin at the desired site. In some embodiments, the targeting domain and / or the retention domain is linked to the interleukin via a linker and is not cleaved at the site of action (e.g., by a protease specific for inflammation or cancer), causing the cytokine to remain at the desired site.

[0134] The antigen to be selected is, in some cases, expressed on the surface of the diseased cells or tissues, such as tumors or cancer cells. Antigens useful for targeting and retention in tumors include, but are not limited to, EpCAM, EGFR, HER-2, HER-3, c-Met, FOLR1, and CEA. The pharmaceutical compositions disclosed herein also include proteins containing two targeting domains 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.

[0135] Suitable targeting domains and / or retention domains include antibody and its fragments such as antigen-binding domains, e.g., polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, single-chain variable fragments (scFv), single-domain antibodies (variable domains of heavy-chain variable domain (VH), light-chain variable domain (VL) and camelid nanobody (VHH), etc.), dAb, etc. Other suitable antigen-binding domains include non-immunoglobulin proteins that mimic the binding and / or structure of antibodies, e.g., anticalins, affilins, afibody molecules, affimers, affitins, alphabodies, avimers, DARPin, fynomers, knotted domain peptides, monobodies, and binding domains based on other engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin, and CTLA4. Further examples of antigen-binding polypeptides include ligands for a desired receptor, ligand-binding portions of receptors, lectins, and peptides that bind or associate with one or more target antigens.

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

[0137] The antigen for targeting and / or holding 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, DLL3, EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6, HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16, NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1, SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, WT1.

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

[0139] The antigen of the target and / or the retention can be a cell surface molecule such as a protein, lipid, or polysaccharide. In some embodiments, the antigen of the target and / or the retention is on tumor cells, virus-infected cells, bacteria-infected cells, damaged red blood cells, arterial plaque cells, inflamed tissue cells, or fibrotic tissue cells. The antigen of the target and / or the retention can include an immune response modulator. Examples of immune response modulators include, but are not limited to, granulocyte macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G-CSF), interleukin 2 (IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.

[0140] The antigen of the target and / or the retention can 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), IFN-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 receptor, 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 thereto.

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

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

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

[0144] Proteases known to be associated with diseased cells or tissues include, but are not limited to, serine proteases, cysteine proteases, aspartic proteases, threonine proteases, glutamic acid proteases, metalloproteases, asparagine peptide lyases, serum proteases, cathepsins, 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 protease, trypsin-like protease, elastase-like protease, subtilisin-like protease, actinidin, bromelain, calpain, caspase, caspase-3, Mirl-CP, papain, HIV-1 protease, HSV protease, CMV protease, chymosin, renin, pepsin, matriptase, legumain, plasminopepsin, 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 sequences encoded by the chimeric nucleic acid sequences provided herein can be selected, for example, from the group consisting of prostate-specific antigen (PSA), matrix metalloprotease (MMP), A disintegrin and metalloprotease (ADAM), plasminogen activator, cathepsin, caspase, tumor cell surface protease, and elastase. MMP can be, for example, matrix metalloprotease 2 (MMP2) or matrix metalloprotease 9 (MMP9).

[0145] Table 1 shows proteases useful in the methods disclosed herein, and exemplary proteases and their cleavage sites are shown in Table 1a. Table 1. Proteases related to inflammation and cancer

Table 1-1

Table 1-2

Table 1-3

Table 1a-1

Table 1a-2

[0146] This specification provides a pharmaceutical composition comprising a polypeptide sequence. As with all peptides, polypeptides, and proteins (including fragments thereof), it is understood that additional modifications can occur in the amino acid sequence of chimeric polypeptides (amino acid sequence variants) that do not modify the properties or functions of the peptide, polypeptide, or protein. Such modifications include conservative amino acid substitutions and are discussed in more detail below.

[0147] The compositions provided herein have a desired function. The composition comprises at least an IL-2 polypeptide, a blocking moiety, such as a steric blocking polypeptide, etc., as well as an optional serum half-life extending element, and an optional target-directed polypeptide, and further one or more linkers connecting each polypeptide in the composition. A first polypeptide, such as an IL-2 mutant protein, is provided as an active substance. The blocking moiety is provided to block the activity of the interleukin. The 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 substance. Optionally, the blocking moiety blocks the activity of the first polypeptide by binding to the interleukin polypeptide. In some embodiments, the blocking moiety, such as 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 release the full activity of the cytokine at the desired site.

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

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

[0150] Cleavage and inducibility As described herein, in the fusion protein, the activity of the cytokine polypeptide is attenuated, and cleavage by a protease at the desired site of activity, such as within the tumor microenvironment, releases a form of the cytokine from a fusion protein that is much more active as a cytokine receptor agonist than the fusion protein. For example, the cytokine receptor activation (agonist) activity of the fusion polypeptide may be at least about 10-fold, at least about 50-fold, at least about 100-fold, at least about 250-fold, at least about 500-fold, or at least about 1000-fold less than the cytokine receptor activation 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 the same amino acids as the cytokine polypeptide, contains substantially no 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 sequences, such as tags or short sequences, to aid in expression and / or purification.

[0151] In other examples, the cytokine receptor activating (agonist) activity of 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 about 1000-fold less than the cytokine receptor activating activity of the polypeptide containing the cytokine polypeptide produced by cleavage of the protease-cleavable linker in the fusion protein. In other words, the cytokine receptor activating (agonist) activity of the polypeptide containing the cytokine polypeptide produced by cleavage of the 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.

[0152] Polypeptide substitution The polypeptides described in this specification can include components (such as cytokines, blocking moieties) having the same amino acid sequence as the corresponding naturally occurring proteins (such as IL-2, IL-15, HSA) as long as the desired function is maintained, or can have an amino acid sequence different from that of the naturally occurring proteins. It is understood that one way to define any known modifications and derivatives or those that can occur of the disclosed proteins and the nucleic acids encoding them is by defining sequence variants in terms of identity to a specific known reference sequence. Specifically, polypeptides and nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the chimeric polypeptides provided herein are disclosed. For example, polypeptides or nucleic acids having at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence of any of the nucleic acids or polypeptides described herein are provided. Those skilled in the art can readily understand the method for determining the identity of two polypeptides or two nucleic acids. For example, the identity can be calculated after aligning the two sequences such that the identity is at its highest level.

[0153] Another way to calculate identity can be performed by published algorithms. Optimal alignment of arrays for comparison can be achieved by the local identity algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), the identity alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), by implementations of these algorithms on a computer (GAP, BESTFIT, FASTA, and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by inspection and confirmation.

[0154] The same type of identity for nucleic acids can be obtained 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 hereby incorporated by reference for at least materials related to nucleic acid alignment. It is understood that any of these methods can typically be used and, in some cases, the results of these various methods may differ, but one of ordinary skill in the art will understand that when identity is found by at least one of these methods, the sequence is said to have the described identity and is disclosed herein.

[0155] Protein modification includes modification of the amino acid sequence. Modifications in the amino acid sequence can occur naturally as allelic variations (e.g., due to gene polymorphisms), can occur due to environmental influences (e.g., exposure to ultraviolet light), or can be created by human intervention such as induced point mutants, deletion mutants, insertion mutants, and substitution mutants (e.g., by mutagenesis of cloned DNA sequences). These modifications can result in changes in the amino acid sequence, provide silent mutations, modify restriction sites, or provide other specific mutations. Modifications of the amino acid sequence typically fall into one or more of three types: substitution modifications, insertion modifications, or deletion modifications. Insertions include fusions at the amino and / or carboxyl termini, as well as in-sequence insertions of single or multiple amino acid residues. Insertions are usually smaller than insertions by fusion at the amino or carboxyl terminus, e.g., insertions of about 1 to 4 residues. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, about 2 to 6 or fewer residues are deleted at any one site within the protein molecule. Amino acid substitutions are typically single residue substitutions, but can occur at several different positions at once, insertions are usually on about 1 to 10 amino acid residues, and deletions are in the range of about 1 to 30 residues. Deletions or insertions are preferably made in adjacent pairs, i.e., deletions of 2 residues or insertions of 2 residues. Combinations of substitutions, deletions, insertions, or any combination thereof may be combined to reach the final construct. Mutations should not place sequences out of the reading frame and preferably should not create complementary regions that can generate secondary mRNA structures. Substitution modification is a modification in which at least one residue is removed and a different residue is inserted in its place. Such substitutions are generally made according to Table 2 below and are called conservative substitutions. Table 2. Exemplary Amino Acid Substitutions

Table 2-1

Table 2-2

[0156] Modifications, including specific amino acid substitutions, are made by known methods. For example, a modification is made by site-directed mutagenesis of the nucleotides of the DNA encoding the polypeptide, whereby DNA encoding the modification is generated, and then the DNA is expressed in recombinant cell culture. Techniques for making substitution mutations at a given site in DNA having a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis.

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

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

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

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

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

[0162] In some embodiments, two cytokines are included in the same construct, at least one of which is IL-2. In some embodiments, each cytokine is connected to two blocking domains (a 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 the pharmacokinetic properties.

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

[0164] Preferred half-life extension elements for use in fusion proteins are human serum albumin (HSA), an antibody or antibody fragment that binds to serum albumin (e.g., scFV, dAb), human or humanized IgG, or 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 to serum albumin, an antibody that binds to a cytokine and prevents or inhibits activation of 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 abundant on the surface of cancer cells, such as EpCAM, FOLR1, and fibronectin.

[0165] Therapeutic methods and pharmaceutical compositions Further provided is a method of treating a subject having or at risk of developing a disease or disorder such as a proliferative disease, a neoplastic disease, an inflammatory disease, an immune disorder, an autoimmune disease, an infectious disease, a viral disease, an allergic reaction, a parasitic reaction, or graft-versus-host disease. A method of 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 mutant protein thereof that is activated at the site of inflammation or tumor. In one embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutant protein thereof, and a serum half-life extension element. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or 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 mutant protein thereof. In another embodiment, the chimeric polypeptide comprises a cytokine polypeptide, a fragment or mutant protein thereof, a blocking moiety, and a serum half-life extension element.

[0166] Inflammation is part of a complex biological response of body tissues to harmful stimuli such as pathogens, damaged cells, irritants, etc., and is a defensive reaction involving immune cells, blood vessels, and molecular mediators. The function of inflammation is to eliminate the initial cause of cell injury, remove necrotic cells and necrotic tissues damaged by the original injury and the inflammatory process, and initiate tissue repair. Inflammation can occur due to infection, occur as a symptom, or occur as a disease, such as cancer, atherosclerosis, allergy, myopathy, HIV, obesity, or autoimmune disease. Autoimmune disease is a chronic pathological condition 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.

[0167] The pharmaceutical composition can include one or more protease-cleavable linker sequences. The linker sequences serve to provide flexibility between the polypeptides such that each polypeptide can inhibit the activity of the first polypeptide. The linker sequences can be positioned between any or all of the cytokine polypeptide, its fragment or mutant protein, the blocking moiety, and the serum half-life extension element. Optionally, the composition includes two, three, four, or five linker sequences. The linker sequences, two, three, or four linker sequences can be the same or different linker sequences. In one embodiment, the linker sequence includes GGGGS (SEQ ID NO: 132), GSGSGS (SEQ ID NO: 133), or G(SGGG)2SGGT (SEQ ID NO: 134). In another embodiment, the linker includes a protease-cleavable sequence selected from the group consisting of HSSKLQ (SEQ ID NO: 25), GPLGVRG (SEQ ID NO: 128), IPVSLRSG (SEQ ID NO: 129), VPLSLYSG (SEQ ID NO: 130, and SGESPAYYTA (SEQ ID NO: 131).

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

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

[0170] Furthermore, a method of treating a subject having or at risk of developing cancer is provided. The method typically comprises administering to a subject in need thereof an effective amount of a chimeric polypeptide (fusion protein) disclosed herein, which is 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, a 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.

[0171] The method further includes administering one or more additional agents for treating cancer, such as chemotherapeutic agents (e.g., adriamycin, cerubidine, bleomycin, alkeran, velban, oncovin, fluorouracil, thiotepa, methotrexate, bisantrene, noantrone, thiguanine, cytaribine, procarabazine), tumor immunotherapy agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47, anti-GD2), cell therapy agents (e.g., CAR-T, T cell therapy), oncolytic viruses, etc.

[0172] Provided herein are pharmaceutical formulations or compositions containing a chimeric polypeptide and a pharmaceutically acceptable carrier. The compositions provided herein are suitable for administration in vitro or in vivo. A pharmaceutically acceptable carrier means a material that is not biologically or otherwise undesirable, i.e., such material does not cause undesirable biological effects or interact in a harmful manner with the other components of the pharmaceutical formulation or composition in which it is contained when administered to a subject. The carrier is selected to minimize degradation of the active ingredient and to minimize any harmful side effects in the subject.

[0173] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy, 21 stIt is described in Edition, David B. Troy, ed., Lippicott Williams & Wilkins (2005). Typically, an appropriate amount of a pharmacologically acceptable salt is used in the formulation to make the formulation isotonic, although if desired, the formulation can be hypertonic or hypotonic. Examples of pharmacologically acceptable carriers include, but are not limited to, sterile water, physiological saline, buffer solutions such as Ringer's solution, and dextrose solutions. The pH of the solution is generally from about 5 to about 8 or from about 7 to 7.5. Other carriers include sustained-release preparations such as the semipermeable matrix of a solid hydrophobic polymer containing the immunogenic polypeptide. The matrix is in the form of a shaped article such as, for example, a film, liposome, or microparticle. A particular carrier may be more preferred depending, for example, on the route of administration and the concentration of the composition being administered. The carrier is suitable for the administration of the chimeric polypeptide or the nucleic acid sequence encoding the chimeric polypeptide to a human or other subject.

[0174] The pharmaceutical formulation or composition is administered in several ways depending on whether local or systemic treatment is desired and depending on the area being treated. The composition is administered via any of several routes of administration, including, but not limited to, topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, transdermal, intrahepatic, intracranial, spray / inhalation, or by instillation via bronchoscopy. In some embodiments, the composition is administered locally (not systemically), such as intratumorally, intra-articularly, intrathecally, etc.

[0175] 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, etc., and injectable organic esters such as ethyl oleate, etc. Aqueous carriers include water, alcoholic solutions / aqueous solutions, emulsions or suspensions, and include physiological saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous vehicles include liquids and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases are optionally present.

[0176] Preparations for topical administration include ointments, lotions, creams, gels, instillations, suppositories, sprays, solutions and powders. Conventional pharmaceutical carriers, aqueous bases, powder bases or oily bases, thickening agents, etc. are optionally necessary or desirable.

[0177] Compositions for oral administration include powders or granules, suspensions or solutions dissolved in water or non-aqueous media, capsules, sachets or tablets. Thickening agents, flavoring agents, diluents, emulsifying agents, dispersion aids or binders are optionally desirable.

[0178] 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 an expression vector. These methods and compositions can be broadly classified into two types: viral delivery systems and non-viral delivery systems. Such methods are well known in the art and can be readily adapted for use with the compositions and methods described herein. Such compositions and methods can be used to transfect or transduce cells in vitro or in vivo, for example, to produce cell lines that express, preferably secrete, the encoded chimeric polypeptide, or for the therapeutic delivery of nucleic acids to a subject. The components of the chimeric nucleic acids disclosed herein are typically operably linked in-frame so as to encode a fusion protein.

[0179] As used herein, a plasmid or viral vector is a substance that transports the disclosed nucleic acid into cells in an undegraded state and contains a promoter that results in the expression of a nucleic acid molecule and / or polypeptide in the target cell. Viral vectors include, for example, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poliovirus, Sindbis, and other RNA viruses, including those having an HIV backbone among these viruses. Also preferred are any viral families that share the characteristics 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), and are incorporated herein by reference for the vectors and methods for their production. The construction of replication-deficient adenoviruses has been described (Berkner et al., J. Virol. 61:1213-20 (1987), Massie et al., Mol. Cell. Biol. 6:2872-83 (1986), Haj-Ahmad et al., J. Virol. 57:267-74 (1986), Davidson et al., J. Virol. 61:1226-39 (1987), Zhang et al., BioTechniques 15:868-72 (1993)). The advantages and uses of these viruses as vectors are that they can replicate within the first infected cells but are limited in their ability to spread to other cell types because they cannot form new infectious virus particles. Recombinant adenoviruses have been shown to achieve high efficiency after direct in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma, and many other tissue sites. Other useful systems include, for example, replication vaccinia virus vectors and host-restricted non-replicating vaccinia virus vectors.

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

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

[0182] The provided polypeptide can be delivered by envelope aggregates. Methods for producing and using envelope aggregates are described in International Publication No. WO2006 / 110728.

[0183] Non-viral delivery methods can include expression vectors containing nucleic acid molecules and nucleic acid sequences encoding polypeptides, where the nucleic acid is operably linked to expression control sequences. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, artificial chromosomes, BACs, YACs, or PACs. A number of vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clonetech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). Vectors typically contain one or more regulatory regions. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response sequences, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription start sites, termination sequences, polyadenylation sequences, and introns. Such vectors can also be used to produce chimeric polypeptides by expression in suitable host cells such as CHO cells.

[0184] Preferred promoters for controlling transcription from vectors in mammalian host cells can be obtained from a variety of sources, for example, from the genomes of viruses such as polyomavirus, simian virus 40 (SV40), adenovirus, retrovirus, hepatitis B virus, 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., a CMV promoter fused to the β-actin promoter). Of course, promoters from the host cell or related species are also useful herein.

[0185] An enhancer generally refers to a DNA sequence whose distance from the functional transcription start site is not constant and can be either 5' or 3' relative to the transcription unit. Furthermore, enhancers can be within introns or even within the coding sequence itself. They are usually 10 - 300 base pairs (bp) in length and function in cis. Enhancers typically function to increase transcription from a nearby promoter. 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 typically, enhancers from eukaryotic viruses are used for general expression. Preferred examples are the SV40 enhancer on the late side of the origin of replication, the cytomegalovirus immediate-early promoter enhancer, the polyoma enhancer on the late side of the origin of replication, and the adenovirus enhancer.

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

[0187] Vectors 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, on a cell. A marker product is used to determine whether the vector has been delivered to the cell and, after delivery, whether it is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such a selectable marker is successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive when placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. Further, an expression vector can include a tag sequence designed to facilitate the manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as sequences of GFP, glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, Conn.), etc., 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.

[0188] As used herein, the terms peptide, polypeptide, or protein are used broadly to mean two or more amino acids linked by peptide bonds. Proteins, peptides, and polypeptides 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 particular size or number of amino acids constituting a molecule, and that the peptides of the invention can contain from a few to several more amino acid residues. Throughout, the subject can be a vertebrate, more specifically, a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), bird, reptile, amphibian, fish, and any other animal. Such terms do not imply a particular age or sex. Thus, both male and female, adult and neonatal subjects are intended to be included. As used herein, patient or subject may be used interchangeably and can refer to a subject having a disease or disorder (e.g., cancer). The terms patient or subject include human subjects and veterinary subjects.

[0189] A subject at risk of developing a disease or disorder can have a genetic predisposition to that disease or disorder, e.g., have a family history, or have a genetic mutation that causes that disease or disorder, or exhibit early signs or symptoms of that disease or disorder. A subject currently having a disease or disorder has one or more symptoms of that disease or disorder and may be diagnosed with that disease or disorder.

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

[0191] In accordance with 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 the desired physiological response. The effective amount and schedule for administering an agent may be determined empirically, and making such determination is within the skill of the art. The dosage range for administration is high enough to produce the desired effect such that one or more symptoms of the disease or disorder are affected (e.g., reduced or delayed). The dosage should not be so large as to cause substantial adverse side effects such as unwanted cross-reactions, anaphylactic reactions, etc. Generally, the dosage varies depending on age, condition, gender, type of disease, degree of the 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. The dosage can be adjusted by an individual physician if there are contraindications. The dosage can vary and can be administered in one or more doses daily for several days or for one or several days. References to a handbook can be found for the appropriate dosage for a given class of pharmaceuticals.

[0192] As used herein, the terms "treat," "treating," or "treatment" refer to a method of affecting a disease or condition or reducing the symptoms 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 the symptoms of the disease or condition. For example, a method for treating a disease is considered a treatment if there is a 10% reduction in one or more symptoms of the disease in the subject compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any reduction rate between 10% and 100% compared to the level in nature or the control. It is understood that treatment does not necessarily refer to the cure or complete elimination of the disease, condition, or the symptoms of the disease or condition.

[0193] As used herein, the terms "prevent," "preventing," and "prevention" with respect to a disease or disorder refer to an act, such as the administration of a chimeric polypeptide or a nucleic acid sequence encoding a chimeric polypeptide, which is performed before or substantially simultaneously with the onset of one or more symptoms of the disease or disorder in a subject, and which inhibits or delays the onset or worsening of one or more symptoms of the disease or disorder. As used herein, references to a decrease, reduction, or inhibition include a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater change compared to a control level. Such terms can include, but do not necessarily include, complete elimination.

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

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

[0196] Furthermore, variants can be created that selectively modify the affinity for the CD25 chain compared to native IL-2.

[0197] IL-2 can be engineered to produce variants that either bind generally to the IL-2R complex or specifically bind to the IL-2Rα subunit with an affinity different from that of the corresponding wild-type IL-2 or currently available variants (referred to as C125S, in which the cysteine residue at position 125 is replaced by a serine residue).

[0198] Accordingly, the present invention features a mutant interleukin-2 (IL-2*) polypeptide having an amino acid sequence that is at least 80% identical (e.g., 85%, 87%, 90%, 95%, 97%, 98%, or 99% identical) to wild-type IL-2 and that binds more avidly to the IL-2 trimeric receptor than to the dimeric IL-2 receptor, as 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 the affinity with which wild-type IL-2 binds to IL-2Rα. The amino acid sequence within the mutant IL-2 polypeptide can contain (or consist of) one or more amino acid substitutions that can be considered conservative or non-conservative substitutions, thereby differing from SEQ ID NO:1 (UniProtKB accession number P60568). Non-naturally occurring amino acids can also be incorporated. Alternatively, or in addition, the amino acid sequence can differ from SEQ ID NO:1 (which can be considered a "reference" sequence) by containing one or more amino acid residues, as well as by addition and / or deletion. More specifically, the amino acid sequence can differ from that of SEQ ID NO:1 by mutations 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 combinations thereof) of SEQ ID NO:1. As described above, only one of these positions can be modified, or 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more (including all up to the maximum) positions can be modified. For example, the amino acid sequence can differ from SEQ ID NO:1 at positions 69 and 74 and can further differ at one or more of positions 30, 35, and 128.The amino acid sequence can also differ from SEQ ID NO: 2 (disclosed in US7569215, which is incorporated herein by reference) at one set of positions selected from the following: (a) positions 64, 69, and 74; (b) positions 69, 74, and 101; (c) positions 69, 74, and 128; (d) positions 30, 69, 74, and 103; (e) positions 49, 69, 73, and 76; (f) positions 69, 74, 101, and 133; (g) positions 30, 69, 74, and 128; (h) positions 69, 74, 88, and 99; (i) positions 30, 69, 74, and 128; (j) positions 9, 11, 35, 69, and 74; (k) positions 1, 46, 49, 61, 69, and 79; (l) positions 48, 68, 71, 90, 103, and 114; (m) positions 4, 10, 11, 69, 74, 88, and 133; (n) positions 15, 30, 31, 35, 48, 69, 74, and 92; (o) positions 30, 68, 69, 71, 74, 75, 76, and 90; (p) positions 30, 31, 37, 69, 73, 74, 79, and 128; (q) positions 26, 29, 30, 54, 67, 69, 74, and 92; (r) positions 8, 13, 26, 30, 35, 37, 69, 74, and 92; and (s) positions 29, 31, 35, 37, 48, 69, 71, 74, 88, and 89. Except for mutations at these positions, the amino acid sequence of the mutant IL-2 polypeptide can be identical to SEQ ID NO: 1 at other points. For certain substitutions, the amino acid sequence can differ from SEQ ID NO: 1 by having one or more of 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, E68D, V69A, N71T, N71A, N71R, A73V, Q74P, S75P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A, F103S, I114V, I128T, I128A, T133A, or T133N.The nomenclature used here is consistent with that of the scientific literature, where the one-letter designation of the amino acid of the wild-type or reference sequence is followed by its position within the sequence, and then the one-letter designation of the amino acid that replaces it. Thus, A1T represents the substitution of the alanine residue at position 1 by threonine. Other mutant polypeptides within the scope of the present invention include those containing variants of SEQ ID NO: 2 having substitutions at V69 (e.g., A) and Q74 (e.g., P). For example, the amino acid sequences include the following sets of mutations (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, 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 may be included. SEQ ID NO: 2 is disclosed in US7569215 and is incorporated herein by reference as an exemplary IL-2 polypeptide sequence useful in the present invention.

[0199] As described above, any of the mutant IL-2 polypeptides disclosed herein can include the recited sequences, and they can be limited to the recited sequences or, in other cases, can be identical to SEQ ID NO:1. Further, any of the mutant 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.

[0200] The mutant IL-2 polypeptides disclosed herein bind to the IL-2Rα subunit with a K of less than about 28 nM d (e.g., less than about 25 nM, less than about 5 nM, about 1 nM, less than about 500 pM, or less than about 100 pM). Specifically, the mutant IL-2 polypeptide can 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 can also be expressed as the relative dissociation rate from the IL-2Rα subunit or the IL-2 receptor complex (e.g., the complex expressed on the surface of a cell or otherwise membrane-bound). For example, the mutant IL-2 polypeptide can dissociate at a lower rate, e.g., from IL-2Rα etc., compared to a therapeutic agent using a wild-type polypeptide or IL-2, e.g., IL-2*. Alternatively, affinity can be characterized as the time or average time that the IL-2* polypeptide persists on the surface of a cell expressing, e.g., IL-2R. For example, the IL-2* polypeptide can persist on the receptor for at least about 2-fold, 5-fold, 10-fold, 50-fold, 100-fold, or 250-fold (or more).

[0201] Materials, compositions, and components are disclosed that can be used in, or in combination with, the disclosed methods and compositions, can be used in their preparation, or are products thereof. These and other materials are disclosed herein, and although specific references to each of the various individual and collective combinations and permutations of these compounds may not be expressly disclosed where combinations, subsets, interactions, groups, etc. of these materials are disclosed herein, each is specifically contemplated and understood to be described herein. For example, if a method is disclosed and discussed, and several modifications that can be made to several molecules that include that method are discussed, every combination and permutation of that method, as well as possible modifications, are specifically contemplated unless another meaning is expressly stated. Similarly, any subset or combination of these is also specifically contemplated and disclosed. This concept applies to all aspects of the present disclosure, including but not limited to steps in methods of using the disclosed compositions. Thus, if there are various additional steps that can be performed, each of these additional steps can be performed using any specific method step or combination of method steps of the disclosed method, and each such combination or subset of combinations is to be specifically contemplated and understood to be disclosed.

[0202] Publications cited herein and the materials for which they are cited are specifically incorporated herein by reference in their entirety.

Examples

[0203] The following are examples of the methods and compositions of the present invention. It is understood that various other embodiments may be practiced based on the generalities provided herein.

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

[0205] Example 2: Protease cleavage of the fusion protein by MMP9 protease One skilled in the art will be familiar with methods for setting up protein cleavage assays. 100 μg of the protein in 1× PBS (pH 7.4) was cleaved with 1 μg 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 either used in functional analysis or stored at -80 °C prior to testing. The degree of cleavage was monitored by SDS PAGE using methods well known in the art. As shown in Figure 10, complete cleavage of the fusion protein by MMP9 protease is seen.

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

[0207] Example 4: Protease cleavage of the IL-2 / IL-2Rα / IL-2Rγ chimeric polypeptide results in increased accessibility to antibodies and biologically active IL-2 mutant proteins Using a protease, e.g., PSA, the IL-2 mutant protein fusion protein before and after cleavage is biochemically characterized. Immunoblot analysis shows that the fusion protein can be cleaved by PSA and that there is an increase in the intensity of the predicted low molecular weight cleavage product of approximately 20 kDa that is reactive with an anti-IL-2 antibody after sample treatment with PSA. The degree of cleavage depends on the amount of PSA and the incubation time. Interestingly, analysis of the fusion protein before and after PSA treatment by ELISA revealed an increase in the apparent amount of IL-2 after PSA cleavage. In this experiment, depending on the construct, there was an approximately 2-fold or 4-fold increase in the apparent amount of IL-2 detected using this sandwich ELISA, suggesting that antibody binding is partially inhibited by the intact fusion protein. Aliquots of the same sample were also analyzed after PSA treatment using the CTLL-2 cell line that requires IL-2 for growth and survival, and cell survival could 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 there is an increase in the amount of biologically active IL-2 after PSA cleavage. The increased amount of the IL-2 mutant protein suggests an increase in the predicted low molecular weight cleavage fragment of approximately 20 kDa that is reactive with an anti-IL-2 antibody, an increase in antibody accessibility, and most importantly, an increase in the amount of biologically active IL-2 mutant protein after PSA cleavage.

[0208] Example 5. In Vivo Delivery of Protease-Activated Fusion Protein Results in Reduced Tumor Growth Examine the chimeric polypeptide to determine whether it may have biological effects in vivo. In these experiments, a system is used in which tumor cells injected intraperitoneally first bind and proliferate to the omentum milky spots, a series of organized immune aggregates found in the omentum, rapidly and preferentially (Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). In this system, since the fusion protein can be delivered intraperitoneally multiple times and tumor growth can be analyzed by examining dissociated omental cells, a convenient method for examining the effect of fusion protein treatment on tumor growth is provided. In these experiments, the Colon38 cell line, a rapidly proliferating tumor cell line that expresses both MMP2 and MMP9 in vitro, can be used. The omental tissue usually expresses relatively small amounts of MMP2 and MMP9, but the MMP levels increase when Colon38 tumors are present in the omentum. Using this tumor model, examine the ability of the IL-2 mutant protein fusion protein to affect tumor growth. Inject Colon38 cells intraperitoneally, and after allowing them to bind and proliferate for 1 day, treat them intraperitoneally with the fusion protein daily. On day 7, sacrifice the animals and examine the omentum for tumor growth using flow cytometry and colony formation assays.

[0209] Example 6: Construction of an exemplary activatable IL2 protein targeting CD20 Generation of an activatable IL2 domain An IL-2 polypeptide capable of binding to the CD20 polypeptide present in a tumor or tumor cell is prepared as follows. Generate a nucleic acid to contain (1) a nucleic acid sequence encoding the IL-2 polypeptide sequence and (2) the nucleic acid sequence of one or more polypeptide linkers. 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. The validation assay includes a T cell activation assay using T cells that are responsive to IL-2 stimulation in the presence of protease.

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

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

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

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

[0214] To adapt to growth in suspension, cells are detached from the tissue culture flask and placed in serum-free medium. Cells adapted to suspension are cryopreserved in medium containing 10% DMSO.

[0215] A recombinant CHO cell line that stably expresses a secreted activatable interleukin protein is generated by transfection of suspension-adapted cells. During selection with the antibiotic hygromycin B, the viable cell density is measured twice a week, the cells are centrifuged, and resuspended in fresh selection medium at a maximum density of 0.1×10 6 viable cells / mL. Two to three weeks after selection, a cell pool that stably expresses the activatable interleukin protein is recovered, at which point the cells are transferred to standard medium in a shaking flask. Protein gel electrophoresis or flow cytometry is performed to confirm the expression of the recombinant secreted protein. The stable cell pool is cryopreserved in medium containing DMSO.

[0216] The 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, typically, the cell culture supernatant is recovered with a culture viability of over 75%. Samples are taken from the production culture every other day to evaluate cell density and viability. On the day of recovery, the cell culture supernatant is removed by centrifugation and vacuum filtered prior to further use.

[0217] The protein expression level and product integrity in the cell culture supernatant are analyzed by SDS-PAGE.

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

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

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

[0221] The binding and cross-reactivity of CD20 to + human CD20 tumor cell lines are evaluated. The K D ratio of cross-reactivity is calculated using the K D value determined for CHO cell lines expressing recombinant human antigen or recombinant cynomolgus antigen.

[0222] Example 8: Cytotoxicity Assay The activatable IL2 protein of Example 6 is evaluated in vitro for its mediation of the immune response against its CD20 + target cells.

[0223] Fluorescently labeled CD20 +REC-1 cells (mantle cell lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMCs from random donors or CB15 T cells (standardized T cell line) as effector cells in the presence of the activatable IL2 protein of Example 5 and at least one protease. After incubation at 37°C for 4 hours in a humidified incubator, the release of the fluorescent dye from the target cells into the supernatant is measured with a spectrofluorometer. Target cells incubated without the activatable IL2 protein of Example 1 and target cells completely lysed by the addition of saponin at the end of the incubation are used as negative and positive controls, respectively.

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

[0225] Example 9: Pharmacokinetics of the activatable IL2 protein The activatable IL2 protein of Example 5 is evaluated in an animal study for its half-life of disappearance.

[0226] 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 administration of an IL2 construct that is of equivalent size but lacks the serum half-life extension element. The third and fourth groups each receive administration of an IL2 construct with a serum half-life extension element and a cytokine having CD20 and a serum half-life extension element, both of which are of equivalent size to the activatable interleukin protein. Each test group consists of 5 monkeys. Serum samples are collected at the indicated time points, serially diluted, and the protein concentration is determined using a binding ELISA against CD20.

[0227] Pharmacokinetic analysis is performed using the plasma concentration of the test substance. The group mean plasma data for each test substance, when plotted against time after administration, follow a multi-exponential profile. The data are fitted to a standard two-compartment model using the bolus dose and the first-order rate constant for the distribution and elimination phases. The general equation for obtaining the best fit of the data for intravenous administration 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 into all of the extracellular fluid of the animal, and the second or β-phase portion of the decay curve represents the true plasma clearance. Methods for fitting such equations are well known in the art. For example, A = D / V(α - k21) / (α - β), B = D / V(β - k21) / (α - β), and α and β (when α > β) are the roots of the quadratic equation r 2 +(k12 + k21 + k10)r + k21k10 = 0, and the estimated parameters of V = volume of distribution, k10 = elimination rate, k12 = rate of movement from compartment 1 to compartment 2, k21 = rate of movement from compartment 2 to compartment 1, and D = administered dose are used.

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

[0229] The activatable IL2 protein of Example 5 is expected to have improved pharmacokinetic parameters, such as an increased elimination half-life, compared to a protein lacking a serum half-life extension factor.

[0230] Example 10: Xenograft Tumor Model The activatable IL2 protein of Example 5 is evaluated in a xenograft model.

[0231] Female immunodeficient NOD / scid mice are irradiated at a sublethal dose (2 Gy) and inoculated subcutaneously with 4 × 10 6 Ramos RA1 cells on the right dorsal flank. When the tumors reach 100 - 200 mm 3 in size, the animals are assigned to three treatment groups. Groups 2 and 3 (8 animals each) receive 1.5 × 10 7Inject activated human T cells intraperitoneally. Three days later, the animals in Group 3 are subsequently treated with a total of 9 intravenous administrations (once a day for 9 days) with 50 μg of the activatable interleukin protein of Example 1. Groups 1 and 2 are treated with vehicle only. Measure body weight and tumor volume for 30 days.

[0232] Animals treated with the activatable IL2 protein of Example 5 are expected to have a statistically significant delay in tumor growth compared to their respective vehicle-treated control groups.

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

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

[0235] Example 12: MC38 experiment The MC38 cell line, a rapidly proliferating colon adenocarcinoma cell line that expresses MMP9 in vitro, was used. This tumor model was used to examine the ability of the fusion protein to affect tumor growth. Example 12a: MC38 IL-2 POC Drugs and Treatments:

Table 4-1

Table 4-2

Table 5-1

Table 5-2

Table 6-1

Table 6-2

[0236] Example 13. Conditionally active fusion protein containing a blocking moiety that is a serum albumin binding domain This example describes the production and activity of a fusion protein, preferably a cytokine, that has inducible activity, i.e., is inactive until induced, typically by cleavage of a linker between a blocking portion and an active portion, resulting in separation of the blocking portion from the active portion. The fusion protein contains a single antibody variable domain (dAb) that binds to serum albumin via a CDR loop and binds to an active portion (here, an anti-CD3 scFV) via one or more non-CDR loops (e.g., the C loop). The serum albumin-binding blocking portion is functionally linked to the active portion via a protease-cleavable linker, and the active portion is functionally linked to a target-directed domain (here, an anti-epidermal growth factor receptor (EGFR) dAb or an anti-prostate specific membrane antigen (PSMA) dAb) via a linker that is not cleavable by protease. These fusion proteins can be administered as inactive proteins that are activated upon cleavage of the protease-cleavable linker, followed by release of the inhibitory albumin-binding domain. The anti-CD3 scFV in the fusion protein is an alternative to 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 mutant protein thereof, a target-directed domain, and an albumin-binding dAb that also binds to and inhibits the cytokine or a functional fragment or mutant protein thereof can be prepared using the methods described and exemplified herein. Both steric masking of the cytokine (due to proximity of the cytokine to the bound serum albumin) and specific masking of the cytokine (by binding via a non-CDR loop (e.g., the C loop) to the cytokine) can be provided by an anti-serum albumin dAb that binds to and inhibits the activity of the desired cytokine or a functional fragment or mutant protein thereof. The anti-serum albumin dAb that binds to and inhibits the activity of the desired cytokine or a functional fragment or mutant protein thereof can be obtained using appropriate methods, e.g., by introducing amino acid sequence diversity into a non-CDR loop (e.g., the C loop) of the anti-serum albumin-binding dAb and screening for binding to the desired cytokine, etc.For selection, any suitable method can be used, such as phage display. For example, an exemplary antiserum albumin dab that can be used has the following sequence. The amino acid sequence within the C-loop (bold underlined part) is diversified (e.g., randomized), and the resulting dAb can be screened for binding to serum albumin via CDR interaction and binding to cytokines via non-CDR loop interaction. If desired, the amino acid sequence of a known cytokine-binding peptide can be introduced into the C-loop.

Chemical formula

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

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

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

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

[0241] B. ProTriTAC exhibits potent protease-dependent antitumor activity in a murine tumor xenograft model In HCT116 subcutaneous xenograft tumors mixed with expanded human T cells in immunodeficient NCG mice, ProTriTAC was evaluated for its in vivo antitumor activity. Specifically, on day 0, for each mouse, 5 × 106 HCT116 cells were mixed with 2.5 × 106 expanded T cells. Administration of ProTriTAC was started the next day and performed by intraperitoneal injection on a schedule of once a day × 10. At the indicated times, tumor volume measurements were obtained using caliper measurements and calculated using the formula V = (length × width × width) / 2.

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

[0243] Stability evaluation The fusion protein purified into two formulations was further equally divided into sterile tubes and subjected to 5 freeze-thaw cycles, each consisting of -80°C and room temperature for more than 1 hour, or incubated at 37°C for 1 week to stress. The stressed samples were evaluated for concentration and turbidity by UV spectrophotometry using a UV-transparent 96-well plate (Corning 3635) with SpectraMax M2 and SoftMaxPro software (Molecular Devices), SDS-PAGE, and analytical SEC, and compared with the same analysis for unstressed control samples. The overlay of chromatograms by analytical SEC of control and stressed samples for a single exemplary ProTriTAC molecule purified from 293 host cells is shown below.

[0244] The results indicate that ProTriTAC was produced at a yield equivalent to that of normal TriTAC from CHO stable pools, and that the protein remained stable after repeated freeze-thaw and after 1 week at 37°C.

[0245] D. Demonstration of Functional Masking and Stability of ProTriTAC In Vivo in a 3-Week Cynomolgus Pharmacokinetic Study A single dose of PSMA-directed ProTriTAC (SEQ ID NO: 119), non-cleavable ProTriTAC (SEQ ID NO: 120), non-masking / non-cleavable TriTAC (SEQ ID NO: 123), and protease-activated ProTriTAC (SEQ ID NO: 121) mimicking the active drug was administered to cynomolgus monkeys by intravenous injection at 0.1 mg / kg. Plasma samples were collected at the indicated time points. The concentration of ProTriTAC was determined using a ligand-binding assay with biotinylated recombinant human PSMA (R&D systems) and a sulfo-tagged anti-CD3 idiotypic antibody clone 11D3 used 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.

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

Equation

Equation

[0247] The clearance rates of the prodrug, active drug, and non-cleavable prodrug control (k c,NCLV ) were empirically determined in cynomolgus monkeys. To estimate the rate of prodrug activation in the circulating blood, the difference in clearance rates between the cleavable and non-cleavable prodrugs was assumed to result only from non-specific activation in the circulating blood. Thus, the rate at which the prodrug is converted to the active drug in the circulating blood was estimated by subtracting the clearance rate of the cleavable prodrug from the clearance rate of the non-cleavable prodrug.

Equation

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

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

[0250] The amino acid sequences of the fusion proteins used in Example 13 are shown as SEQ ID NOs: 116 to 123.

[0251] As an example, the fusion protein constructs are shown in detail in Table 3. In Table 3, "L" is an abbreviation for "linker" and "cleav.link." is an abbreviation for "cleavable linker". Other abbreviations: "mIFNg" indicates mouse interferon gamma (IFNg), "hAlbumin" indicates human serum albumin (HSA), and "mAlbumin" indicates mouse serum albumin. Table 3: Sequence Listing of Constructs

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 7-1

Table 7-2

Table 7-3

Table 7-4

Table 7-5

Table 7-6

Table 7-7

Table 7-8

Table 7-9

Table 7-10

Table 7-11

Table 7-12

Table 7-13

Table 7-14

Table 7-15

Table 7-16

Table 7-17

Table 7-18

Table 7-19

[0252] The entire disclosures of all patents and non - patent publications cited in this specification are hereby incorporated by reference in their entirety for all purposes.

[0253] Other embodiments The above disclosure may include a plurality of distinct inventions having independent utility. Each of these inventions has been disclosed in its preferred form(s), but since numerous variations are possible, the specific embodiments 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 combination components of the various elements, features, functions, and / or characteristics disclosed herein. The following claims particularly point out certain combinations and combination components that are considered novel and non - obvious. Inventions embodied in other combinations and combination components of features, functions, elements, and / or characteristics may be claimed in this application, an application claiming priority from this application, or a related application. Such claims are also considered to be included in the subject matter of the inventions of the present disclosure, whether they are directed to different inventions, or the same invention, and whether the scope is broader, narrower, equal to, or different from the original claims. The present invention provides, for example, the following items. (Item 1) Formula [A] - [L1] - [B] - [L2] - [D] or [A] - [L1] - [D] - [L2] - [B] or [D] - [L2] - [B] - [L1] - [A] or [B] - [L2] - [D] - [L1] - [A] or [D] - [L1] - [B] - [L1] - [A] or [B] - [L1] - [D] - [L1] - [A] [wherein A is an interleukin - 2 (IL - 2) polypeptide, B is an in - vivo half - life extending element, L1 and L2 are each independently a polypeptide linker, L1 is a protease-cleavable polypeptide linker, L2 is an optionally protease-cleavable polypeptide linker, D is a fusion polypeptide which is an IL-2 blocking moiety] The fusion polypeptide has attenuated IL-2 receptor activation activity, and the IL-2 receptor activation activity of the fusion polypeptide is at least about 10-fold lower than the IL-2 receptor activation activity of the polypeptide containing the IL-2 polypeptide produced by cleavage of the protease-cleavable linker L1. Said fusion polypeptide. (Item 2) a) interleukin 2 (IL-2) polypeptide [A], b) an IL-2 blocking moiety [D], and c) a protease-cleavable polypeptide linker [L], a fusion polypeptide comprising at least one of each of The IL-2 polypeptide and the IL-2 blocking moiety are functionally linked by the protease-cleavable polypeptide linker, the fusion polypeptide has attenuated cytokine receptor activation activity, and the cytokine receptor activation activity of the fusion polypeptide is that of the protease-cleavable linker. Said fusion polypeptide, which is at least about 10-fold lower than the cytokine receptor activation activity of the polypeptide containing the cytokine polypeptide produced by cleavage. (Item 3) The agonist activity of the IL-2 polypeptide containing the fragment of the cleaved polypeptide is increased by at least about 50-fold compared to the uncleaved fusion polypeptide, the fusion polypeptide according to item 1 or item 2. (Item 4) The agonist activity is evaluated using a CTLL-2 proliferation assay, a phospho STAT ELISA, or a HEK Blue reporter cell assay, the fusion polypeptide according to any one of items 1 to 3. (Item 5) The non-cleavable fusion polypeptide binds to interleukin-2 receptor alpha (IL-2Rα) in substantially the same manner as naturally occurring IL-2, and is the fusion polypeptide according to any one of items 1 to 4. (Item 6) The blocking portion inhibits the activation of interleukin-2 receptor alpha / beta / gamma (IL-2Rαβγ) and interleukin-2 receptor beta / gamma (IL-2Rβγ) by the IL-2 polypeptide of the non-cleavable fusion polypeptide, and is the fusion polypeptide according to any one of items 1 to 5. (Item 7) Each protease-cleavable linker polypeptide independently contains a sequence that can be cleaved by a protease selected from the group consisting of kallikrein, thrombin, chymase, carboxypeptidase A, cathepsin G, cathepsin L, elastase, PR-3, granzyme M, calpain, matrix metalloprotease (MMP), fibroblast activation protein (FAP), ADAM metalloprotease, plasminogen activator, cathepsin, caspase, tryptase, and tumor cell surface protease, and is the fusion polypeptide according to any one of items 1 to 6. (Item 8) Each protease-cleavable polypeptide independently contains two or more cleavage sites for the same protease, or two or more cleavage sites cleaved by different proteases, or at least one of the protease-cleavable polypeptides contains cleavage sites for two or more different proteases, and is the fusion polypeptide according to any one of items 1 to 7. (Item 9) The IL-2 blocking portion binds non-covalently to the IL-2 polypeptide, and is the fusion polypeptide according to any one of items 1 to 8. (Item 10) The binding by the non-covalent bond is pH-dependent, and is the fusion polypeptide according to item 9. (Item 11) The IL-2 blocking portion is a ligand binding domain or fragment of a cognate receptor for the IL-2, a single domain antibody, Fab, or scFv that binds to the IL-2 polypeptide, or an antibody or antibody fragment (e.g., Fab, single domain antibody, scFv) that binds to the receptor of the IL-2, the fusion polypeptide according to any one of items 1 to 10. (Item 12) The IL-2 blocking portion is also a half-life extending element, the fusion polypeptide according to any one of items 1 to 11. (Item 13) The IL-2 blocking portion sterically blocks the agonist activity of the IL-2 polypeptide, the fusion polypeptide according to any one of items 1 to 8 or 12. (Item 14) The IL-2 blocking portion is human serum albumin, or an antigen-binding polypeptide that binds to human serum albumin, the fusion polypeptide according to item 12 or 13. (Item 15) After the protease-cleavable polypeptide linker is cleaved by a protease, the IL-2 dissociates freely from the IL-2 blocking portion, the fusion polypeptide according to any one of items 1 to 14. (Item 16) The fusion polypeptide binds to IL-2Rα, the fusion polypeptide according to any one of items 1 to 15. (Item 17) The fusion polypeptide according to any one of items 1 to 16, further comprising at least one half-life extending element. (Item 18) The half-life extending element is human serum albumin, or an antigen-binding polypeptide that binds to human serum albumin, the fusion polypeptide according to item 17. (Item 19) The half-life extending element is immunoglobulin Fc, the fusion polypeptide according to item 17. (Item 20) The fusion polypeptide according to item 13, wherein the IL-2 blocking moiety is human serum albumin, human IgG, humanized IgG, sdAb, Fab, and scFv or fragments thereof. (Item 21) The fusion polypeptide according to any one of items 1 to 20, wherein the IL-2 receptor activation is determined using a standard in vitro receptor activation assay and equimolar amounts of the IL-2 polypeptide and the fusion polypeptide based on moles. (Item 22) IL-2 dissociates freely from the IL-2 blocking moiety and / or the half-life extension element after the protease-cleavable sequence is cleaved by a protease, for the fusion polypeptide according to any one of items 1 to 21. (Item 23) The fusion polypeptide according to any one of items 17 to 19, wherein the at least one half-life extension element is one half-life extension element or two half-life extension elements. (Item 24) L2 is a protease-cleavable polypeptide linker, for the fusion polypeptide according to any one of items 1 to 23. (Item 25) L1 is a substrate for a first protease and L2 is a substrate for a second protease, for the fusion polypeptide according to any one of items 1 to 24. (Item 26) The fusion polypeptide according to any one of items 2 to 25, further comprising a tumor-specific antigen-binding peptide. (Item 27) The fusion polypeptide according to item 26, wherein the tumor-specific antigen-binding peptide is linked to the IL-2 polypeptide by a non-cleavable linker. (Item 28) The fusion polypeptide according to item 26, wherein the tumor-specific antigen-binding peptide is linked to the IL-2 polypeptide, the half-life extension element, or the IL-2 blocking moiety by a cleavable linker. (Item 29) The fusion polypeptide according to any one of items 1 to 28, wherein the half-life of the IL-2 polypeptide produced by cleavage of the protease-cleavable linker is equivalent to the half-life of naturally occurring IL-2. (Item 30) The fusion polypeptide according to any one of items 1 to 29, wherein the IL-2 polypeptide comprises a deletion or substitution of a cysteine residue corresponding to Cys125 of SEQ ID NO:. (Item 31) A nucleic acid encoding the polypeptide according to any one of items 1 to 30. (Item 32) A vector comprising the nucleic acid according to item 31. (Item 33) A host cell comprising the vector according to item 32. (Item 34) A method for producing a pharmaceutical composition, comprising culturing the host cell according to item 33 under conditions suitable for expression and collection of the desired polypeptide. (Item 35) A pharmaceutical composition comprising: i) an effective amount of the fusion polypeptide according to any one of items 1 to 30; and ii) a pharmaceutically acceptable additive. (Item 36) 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 30. (Item 37) The fusion polypeptide according to any one of items 1 to 30 for use as a medicament. (Item 38) The fusion polypeptide according to any one of items 1 to 30 for use in a subject in need thereof in the treatment of a tumor. (Item 39) 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 30.

Claims

【Claim 1】 The invention described in the drawings.

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