Interleukin 15 fusion proteins, and compositions and therapeutic methods thereof

Abstract

To provide novel fusion proteins, to provide therapeutic uses thereof, and to provide methods for producing the same.SOLUTION: The present invention provides novel fusion proteins of Interleukin 15 and prodrugs, and compositions and methods of preparation thereof, useful in treating various diseases and disorders (e.g., hyperplasia, solid tumor or hematopoietic malignancy).SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claims and related applications This application claims the benefit of Chinese Application No. 201810420739.6, filed May 4, 2018, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Technical field of the invention The present invention relates generally to novel fusion proteins and their therapeutic uses. More specifically, the present invention provides novel fusion proteins of interleukin-15 and a prodrug, as well as compositions and methods for their preparation, which are useful in the treatment of various diseases and disorders, such as hyperplasias, solid tumors, or hematopoietic malignancies. [Background technology]

[0003] Interleukin-15 (IL15), a 14-15 kDa glycoprotein, is a soluble cytokine first discovered in 1994 (Grabstine et al. 1994 Science 264:965-8). Like interleukin-2, IL15 belongs to the family of four-helix bundle cytokines. The human IL15 gene has been mapped to the q25-35 region of chromosome 4. Mature IL15 consists of 112 amino acids and contains three N-glycosylation sites. IL15 expression is tightly regulated. IL-15 mRNA is found in many tissues and cells, including fibroblasts, muscle cells, keratinocytes, kidney cells, lymphocytes, mast cells, and tumor cells; however, the mature protein is produced primarily by dendritic cells, monocytes, macrophages, and stromal cells, but not by T cells. IL-15 expression is stimulated by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF), interferons, and agonists of toll-like receptors (TLRs) (Marek et al. 2011 Cytokine&Growth Factor Reviews 22:99-108).

[0004] The IL15 receptor (IL15R) belongs to the hematopoietic superfamily. The heterotrimeric IL15R contains α, β (CD122), and γ (CD132, common γ chain, γc) subunits. The β subunit (IL15Rβ) is shared with the IL2 receptor. Human IL15Rα belongs to the type I transmembrane protein family. Both IL2Rα and IL15Rα contain a conserved sushi domain. IL15 shares some functions with IL2, such as promoting the proliferation of T cells and NK cells [3] (Thomas et al. 2006 J of Immunology 177:6072-6080).

[0005] IL15Rα is primarily expressed on dendritic cells (DCs) and monocytes. In most cases, IL15 binds to the receptor in a transpresentation manner. In the transpresentation model, IL15 and IL15Rα are synthesized within the same cell. The IL15 and IL15Rα sushi domains bind to each other with high affinity in the cytoplasm and transport IL-15 to the cell membrane. IL15Rα can then transpresent IL-15 to responding cells, such as T cells and NK cells.

[0006] IL15 exhibits pleiotropic functions in homeostasis and activation of both innate and adaptive immunity, including: (1) IL-15 plays an important role in the activation, proliferation, and survival of CD8+ T cells. (2) IL15 plays an important role in the activation and homeostasis of memory CD8+ T cells. (3) IL15 plays an important role in the development, activation, and proliferation of NK cells and NKT cells. (4) IL15 plays an important role in the production of anti-tumor antibodies. (5) IL-15 plays an important role in the activation, proliferation, and differentiation of DCs in an autocrine model, promoting the expression of MHC II and CD80 / CD86 on DCs and increasing their presentation to CD8+ T cells. (6) IL15 plays an important role in the activation of monocytes and macrophages. (7) IL15 plays an important role in inhibiting AICD, protecting T cells from Treg-mediated inhibition and overcoming tolerance to tumor antigens.

[0007] IL-2 is approved by the FDA for the treatment of metastatic renal cell carcinoma and malignant melanoma. However, the effectiveness of IL-2 as an anticancer therapeutic has been questioned due to its crucial role in maintaining CD4+CD25+ T regulatory cells and in activation-induced cell death (AICD). This process leads to the elimination of stimulated T cells and the induction of T cell tolerance, thereby limiting therapeutic efficacy.

[0008] Unlike IL2, IL15 is not involved in activation-induced cell death (AICD) and the maintenance of regulatory T cells. Therefore, IL15 may have significant advantages over IL2 in the treatment of cancer. A recent report showed that administration of a preformed complex between IL15 and its soluble receptor IL15Rα prolongs the half-life of IL15 and improves the proliferation of T cells and NK cells (Thomas et al. 2006 J of Immunology 177:6072-6080).

[0009] Importantly, a soluble fusion protein of the IL15Rα sushi domain and IL15 linked by a flexible peptide extended the half-life of IL15 and enhanced the proliferation of T cells and NK cells. In mouse B16F10 and DEN-induced HCC tumor models, this fusion protein was able to inhibit tumor growth and suppress tumor metastasis. Furthermore, IL15 showed enhanced antitumor effects or tumor growth suppression in combination studies. (Cheng et al. 2014 J of Hepatology 61:1297-1303; Guo et al. 2017 Cytokine and Growth Factor Reviews 38:10-21.)

[0010] Various side effects are associated with IL15 therapy, including: (1) Induction of a cytokine cascade including TNFα, IL1, IL6, GM-CSF, and proinflammatory cytokines (2) Promoting the proliferation, survival, and metastasis of some tumor cells (3) Activation of autoimmune T cells and involvement in autoimmune diseases (4) Induction of coronary heart disease, and (5) Induction of the expression of the regulatory molecule PD1 / PDL1 is.

[0011] For example, currently available treatments and methods for hyperplasia, solid tumors, or hematopoietic malignancies are inadequate, and there remains an urgent and continuing need for new and improved therapies to effectively treat such diseases and conditions. Summary of the Invention

[0012] The present invention is based in part on the surprising discovery of novel fusion proteins and their therapeutic uses. Disclosed herein are novel fusion proteins of IL15 and its prodrugs, their compositions and methods of preparation, which are useful in treating a variety of diseases and disorders, such as hyperplasias, solid tumors or hematopoietic malignancies.

[0013] In one aspect, the present invention relates generally to a fusion protein comprising a first structural unit: a subunit of interleukin-15 receptor alpha (IL15Rα) or a fragment thereof, a second structural unit: active IL15, a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein, and a first linker segment (L1) covalently linking the first, second, and third structural units, with the first structural unit located at the N-terminus of the fusion protein and the second structural unit located between the first and third structural units.

[0014] In another aspect, the present invention relates generally to a fusion protein comprising a first structural unit: a subunit of interleukin-15 receptor alpha (IL15Rα) or a fragment thereof, a second structural unit: active IL15, a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein, and a first linker segment (L1) covalently linking the first, second, and third structural units, wherein the N-terminus of the fusion protein is the second structural unit, and the first structural unit is located between the second and third structural units.

[0015] In yet another aspect, the present invention relates generally to a fusion protein comprising a first structural unit: an interleukin-15 receptor alpha (IL15Rα) subunit or fragment thereof, a second structural unit: active IL15, a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein, a fourth structural unit: an interleukin-15 receptor beta (IL15Rβ) subunit or fragment thereof, and a first linker segment (L1) covalently linking the first, second, third, and fourth structural units, wherein the N-terminus of the fusion protein is the fourth structural unit, the second structural unit is located between the fourth structural unit and the first structural unit, and the first structural unit is located between the second structural unit and the third structural unit.

[0016] In yet another aspect, the present invention relates to a fusion protein as a whole, comprising: a first structural unit: a subunit of interleukin-15 receptor (IL15R) or a fragment thereof; a second structural unit: active IL15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; a fourth structural unit: a subunit of interleukin-15 receptor beta (IL15Rβ) or a fragment thereof; a first linker segment (L1) covalently linking the first, second, and third structural units; and a second linker segment (L2) covalently linking the two structural units, wherein the first structural unit is located between the second and third structural units, and the N-terminus of the fusion protein is the fourth structural unit.

[0017] In yet another aspect, the present invention relates generally to homodimeric or heterodimeric proteins, including the fusion proteins disclosed herein.

[0018] In yet another aspect, the invention relates to substantially purified proteins, such as fusion proteins or fragments, disclosed herein in their entirety.

[0019] In yet another aspect, the invention relates generally to polynucleotides encoding proteins such as the fusion proteins disclosed herein, or fragments thereof.

[0020] In yet another aspect, the invention generally relates to expression vectors comprising a polynucleotide encoding a protein, such as a fusion protein, or fragment thereof, disclosed herein.

[0021] In yet another aspect, the invention generally relates to pharmaceutical compositions comprising a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0022] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0023] In yet another aspect, the present invention generally relates to a method for treating a disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof disclosed herein, wherein the disease or condition is selected from a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0024] In yet another aspect, the invention relates generally to the use of a protein, such as a fusion protein or fragment thereof, disclosed herein to treat or alleviate a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0025] In yet another aspect, the invention relates generally to the use of polynucleotides encoding proteins such as the fusion proteins disclosed herein or fragments thereof to treat or alleviate a disease or disorder (e.g., hyperplasia, solid tumor, or hematopoietic malignancy).

[0026] In yet another aspect, the invention generally relates to the use of a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or reducing a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0027] In yet another aspect, the invention generally relates to the use of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or reducing a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0028] In yet another aspect, the invention generally relates to cell lines comprising polynucleotides encoding proteins, such as fusion proteins or fragments thereof, as disclosed herein.

[0029] In yet another aspect, the invention generally relates to a process for producing a protein comprising culturing a cell line, which process, in certain embodiments, further comprises purifying or isolating the produced protein, such as a fusion protein or fragment thereof, as disclosed herein.

[0030] In yet another aspect, the invention generally relates to a method of making a protein, the method comprising providing an expression vector encoding a protein, such as a fusion protein or fragment thereof disclosed herein, introducing the expression vector into a host cell, culturing the host cell in a medium under conditions sufficient to express the protein, and purifying the protein from the host cell or medium.

[0031] In yet another aspect, the invention relates generally to isolated proteins produced by the processes disclosed herein. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows schematic diagrams of the structure of fusion proteins. A shows a schematic diagram of the structure of IL15-Fc. B and C show schematic diagrams of two IL15 superagonists (both called super-IL15): RA-IL15-Fc and IL15-RA-Fc. D shows a schematic diagram of the fusion protein RB-IL15-RA-Fc. [Figure 2] FIG. 2 shows exemplary SDS-PAGE electropherograms of the three fusion proteins. [Figure 3] FIG. 3 shows exemplary results of lymphocyte proliferation assays with IL15-Fc and super-IL15. [Figure 4] FIG. 4 shows exemplary results of lymphocyte proliferation assays with RB-IL15-RA-Fc and super-IL15. [Figure 5A] Figure A5 shows exemplary results for the therapeutic effect of Super-IL15 in the A20 tumor model. Exemplary data are shown for the therapeutic effect of Super-IL15 via intratumoral injection. [Figure 5B] Figure 5B shows exemplary results for the therapeutic effect of super-IL15 in the A20 tumor model. Exemplary data for mouse survival after intratumoral and intraperitoneal injection are shown. [Figure 5C]Figure 5C shows exemplary results for the therapeutic effect of super-IL15 in the A20 tumor model. Exemplary data are shown for tumor-cured mice re-challenged with A20 tumor cells. [Figure 6A] Figure 6A shows exemplary results for the therapeutic effect of Super-IL15 in the MC38 tumor model. Exemplary data are shown for the therapeutic effect of Super-IL15 via intratumoral and intravenous injection. [Figure 6B] Figure 6B shows exemplary results for the therapeutic effect of super-IL15 in the MC38 tumor model. Exemplary data for mouse survival after treatment are shown. [Figure 7] FIG. 7 shows exemplary data on the therapeutic effect of super-IL15 in the A20 mouse model at lower doses. [Figure 8A] Figure 8A shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and super-IL15 in the A20 tumor model after intravenous injection. Exemplary tumor growth curves of mice after treatment are shown. [Figure 8B] Figure 8B shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and super-IL15 in the A20 tumor model after intravenous injection. Exemplary levels of cytokines in serum after treatment are shown. [Figure 9A] Figure 9A shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and super-IL15 in a mouse A20 tumor model after intraperitoneal injection. Exemplary survival of mice after treatment is shown. [Figure 9B] Figure 9B shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and super-IL15 in a mouse A20 tumor model after intraperitoneal injection. Exemplary cytokine levels in serum after treatment are shown. [Figure 10]Figure 10 shows exemplary SDS-PAGE electrophoresis of purified human IL15 fusion proteins with or without MMP14 digestion. To emphasize whether L1 or L2 is used as the linker segment attached to RB, RB-IL15-RA-Fc is displayed as RB-L1-15RA-Fc or RB-L2-15RA-Fc. IL15-RA-Fc is displayed as 15RA-Fc. [Figure 11] FIG. 11 shows exemplary results of human IL15 fusion protein activity with or without MMP14 incubation assessed using a HEK-Blue™ IL2 reporter cell assay. DETAILED DESCRIPTION OF THE INVENTION

[0033] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms are intended to have the following meanings unless otherwise indicated by the context in which they are found:

[0034] When trade names are used herein, they include product formulations, generic drugs, and active pharmaceutical ingredients of the trade name product unless the context dictates otherwise.

[0035] Ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0036] As used herein, "at least" a particular value is understood to be that value and all values ​​greater than that value.

[0037] As used herein, "greater than one" is understood as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 100, etc., or any value therebetween.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0039] As used herein, unless specifically stated or clear from the context, the term "about" is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. About is understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the specified value. Unless otherwise indicated by the context, all numerical values ​​provided herein can be modified by the term about.

[0040] As used herein, unless specifically stated otherwise or clear from context, the term "or" is understood to be inclusive.

[0041] When used to define compositions and methods, the term "comprising" is intended to mean including the recited elements but not excluding other elements. When used to define compositions and methods, the term "essentially consisting" is intended to mean that the compositions and methods include the recited elements and exclude any other elements of any essential importance to the compositions and methods. For example, "essentially consisting" refers to the administration of explicitly recited pharmacologically active agents and excludes pharmacologically active agents not explicitly recited. The term "essentially consisting" does not exclude pharmacologically inactive or inactive agents, such as pharmaceutically acceptable excipients, carriers, or diluents. When used to define compositions and methods, the term "consisting of" is intended to mean excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0042] As used herein, the term "agonist" refers to a compound that can combine with a receptor to generate a cellular response. An agonist can be a ligand that directly binds to a receptor. Alternatively, an agonist can bind to a receptor indirectly, for example, by (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise modifying another compound so that the other compound directly binds to the receptor.

[0043] As used herein, the term "antagonist" refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of the agonist or inverse agonist at the receptor. However, an agonist does not affect constitutive receptor activity.

[0044] As used herein, the term "antibody" refers to a molecule capable of binding to an epitope or antigenic determinant. The term is intended to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fab, Fv, single-chain antibodies, and antibodies with one or more immunoglobulin variable chains or CDR domains, as well as bispecific and multispecific antibodies. Antibodies may be derived from any animal. Preferably, antibodies are derived from mammals, such as humans, mice, rabbits, goats, guinea pigs, camels, horses, etc., or other suitable animals. Antibodies can recognize polypeptide or polynucleotide antigens. The term includes, for example, antigen-binding fragments of immunoglobulins, active fragments including the variable and / or constant regions of the heavy chain, the variable and / or constant regions of the light chain, the complementarity-determining regions (CDRs), and the framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, modified antibodies, chimeric antibodies, hybrid antibody molecules, F(ab)2 and F(ab) fragments; Fv molecules (e.g., non-covalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and any functional fragments derived from such molecules that retain specific binding.

[0045] As used herein, the term "antigen" refers to any substance that causes the immune system to generate antibodies or a specific cellular immune response against it. A disease-associated antigen is any substance associated with any disease that causes the immune system to generate antibodies or a specific cellular immune response against it. An antigen can be recognized by the immune system and / or induce a humoral and / or cellular immune response that leads to the activation of B lymphocytes and / or T lymphocytes. An antigen can have one or more epitopes (B cell and / or T cell epitopes). An antigen preferably reacts with its corresponding antibody or TCR in a highly selective manner, and does not react with many other antibodies or TCRs that may be elicited by other antigens. An antigen as used herein may also be a mixture of several individual antigens.

[0046] As used herein, the term "biologically active" or "biologically active" refers to a polypeptide or fragment thereof that possesses a structural, regulatory, or biochemical function of a naturally occurring molecule, or any function related to or associated with a metabolic or physiological process. Biologically active polypeptides or fragments thereof include those that can participate in a biological process or reaction and / or produce a desired effect. Biological activity can include improved desired activity or a reduction in undesired activity. For example, an entity exhibits biological activity if it participates in a molecular interaction with another molecule, has therapeutic value in alleviating a disease state, has preventative value in eliciting an immune response, or has diagnostic and / or prognostic value in determining the presence of a molecule. Biologically active proteins or polypeptides can be naturally occurring or synthesized from known elements, e.g., recombinant or chemical synthesis, and can include heterologous elements.

[0047] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0048] As used herein, the term "cell" refers to any prokaryotic cell, eukaryotic organism, primary or immortalized cell line, any group of such cells, such as a tissue or organ. Preferably, the cells are of mammalian (e.g., human) origin and can be infected by one or more pathogens.

[0049] As used herein, the term "co-administered" refers to the simultaneous presence of two drugs in the blood. The two drugs can be administered simultaneously or sequentially.

[0050] As used herein, the term "co-expressed" means that two different polypeptides are expressed simultaneously in a host cell such that the two polypeptides can interact or bind and form a complex either in the host cell or in the host cell culture medium.

[0051] As used herein, the term "disease" or "disorder" refers to a medical condition, e.g., one that can be identified by symptoms or other distinguishing factors as distinct from a healthy or normal state. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic disorders.

[0052] As used herein, the term "effective amount" of an active agent refers to an amount sufficient to elicit a desired biological response. As will be appreciated by those skilled in the art, the effective amount of the compound of the present invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.

[0053] As used herein, the term "expression of a nucleic acid molecule" refers to the conversion of the information contained in a nucleic acid molecule into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or other type of RNA) or a peptide or polypeptide produced by translation of an mRNA. Gene products also include RNAs modified by processes such as capping, polyadenylation, methylation, and editing; and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0054] As used herein, the term "host cell" refers to an individual cell or cell culture that can be or has been the recipient of any recombinant vector or isolated polynucleotide. Host cells include prokaryotic, eukaryotic, mammalian, avian, insect, plant, or bacterial cells and can be cells of any origin that have been transfected, transformed, transduced, or infected, or that can be used to propagate the nucleic acids described herein. A host cell includes the progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations and / or changes. Host cells include cells transfected or infected in vivo or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention may be referred to as a "recombinant host cell."

[0055] Host cells include, but are not limited to, mammalian, plant, insect, fungal, and bacterial cells. Bacterial cells include, but are not limited to, Gram-positive bacterial cells such as Bacillus, Streptomyces, and Staphylococcus, and Gram-negative bacterial cells such as Escherichia and Pseudomonas. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila cells and Sf9 cells. Plant cells include, inter alia, cells from crop plants such as cereals, medicinal or ornamental plants, or bulbs. Mammalian cells suitable for the present invention include epithelial cell lines (e.g., porcine), osteosarcoma cell lines (e.g., human), neuroblastoma cell lines (e.g., human), epithelial carcinomas (e.g., human), glial cells (e.g., mouse), hepatic cell lines (e.g., monkey), CHO cells (Chinese hamster ovary), COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER., C6, human ECC NTERA-2 cells, mESC line D3 cells, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3 cells, 293T cells, REH cells and MCF-7 cells, and hMSC cells.

[0056] As used herein, the term "Fc" refers to a molecule or sequence comprising the sequence of a non-antigen-binding fragment of a whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of a native Fc is preferably human and can be any immunoglobulin (e.g., IgG1, IgG2). A native Fc is composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide) and non-covalent bonds. The number of intermolecular disulfide bonds between the monomeric subunits of a native Fc molecule ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2).

[0057] As used herein, the term "Fc domain" or "Fc region" refers to a "fragment crystallizable" region of an immunoglobulin heavy chain. Generally, an Fc domain can interact with a second Fc domain to form a dimeric complex. Fc domains can bind to cell surface receptors called Fc receptors and / or proteins of the complement system, or can be altered to reduce or increase their binding activity. Fc domains are derived from IgG, IgA, IgD, IgM, or IgE antibody isotypes and affect immune activities, including opsonization, cytolysis, mast cell degranulation, basophil degranulation, eosinophil degranulation, and other Fc receptor-dependent processes; activation of the complement pathway; and in vivo protein stability.

[0058] "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined herein. As with Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether derived from whole antibodies or produced by recombinant gene expression or other means.

[0059] Fc fusion proteins have been reported that combine the Fc region of IgG with domains from other proteins, such as various cytokines and soluble receptors (e.g., Capon et al. 1989 Nature 337:525-531; Chamow et al. 1996 Trends Biotechnol. 14:52-60; U.S. Patent Nos. 5,116,964 and 5,541,087).

[0060] The use of Fc fusions is known in the art (e.g., U.S. Pat. Nos. 7,754,855; 5,480,981; 5,808,029; WO 7 / 23614; WO 98 / 28427, and references cited therein). Fc fusion proteins can include variant Fc molecules (e.g., as described in U.S. Pat. No. 7,732,570). Fc fusion proteins can be soluble in plasma or can bind to the cell surface of cells bearing specific Fc receptors.

[0061] As used herein, the term "Fc variant" refers to a molecule or sequence that has been altered from a native Fc but still contains a binding site for the salvage receptor, FcRn. International Applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and their interactions with the salvage receptor, and are incorporated herein by reference. Thus, the term "Fc variant" includes molecules or sequences that have been humanized from a non-human native Fc. Additionally, native Fcs contain sites that can be removed because they provide structural features or biological activity not required for the fusion molecules of the present invention. Thus, in certain embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in more detail below.

[0062] As used herein, the term "fusion protein" refers to a polypeptide comprising two or more regions from different or heterologous proteins covalently linked (i.e., "fused") by recombinant, chemical, or other suitable methods. Optionally, the fusion molecule can be fused at one or several sites via a peptide or other linker segment or sequence. For example, one or more peptide linkers can be used to aid in the construction of the fusion protein.

[0063] As used herein, the term "GC content" refers to the percentage of a nucleic acid sequence that contains deoxyguanosine (G) and / or deoxycytidine (C) deoxyribonucleoside, or guanosine (G) and / or cytidine (C) ribonucleoside residues.

[0064] As used herein, the term "high dose" means at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) greater than the highest standard recommended dose of a particular compound for the treatment of a human disease or condition.

[0065] As used herein, the term "immune response" refers to the process by which immune cells are stimulated and / or recruited from the blood to lymphoid and non-lymphoid tissues through a multifactorial process involving distinct adhesion and / or activation steps. Activating conditions result in the release of cytokines, growth factors, chemokines, and other factors, upregulate the expression of adhesion and other activation molecules on immune cells, promote adhesion, morphological changes, and / or extravasation along with chemotaxis through tissues, enhance cell proliferation and cytotoxic activity, stimulate antigen presentation, and provide other phenotypic changes, including the development of memory cell types. Immune response also refers to the activity of immune cells to suppress or modulate the inflammatory or cytotoxic activity of other immune cells. An immune reaction refers to the activity of immune cells in vivo or in vitro.

[0066] The term "identical" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are identical or have an identical specified percentage of amino acid residues or nucleotides (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a designated region (e.g., IL15 or IL15Rα sequences) when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. Such sequences are then said to be "substantially identical." This definition can also refer to, or apply to, the complement of a test sequence. The definition includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the entire length of the amino acid or nucleic acid sequence.

[0067] For sequence comparison, typically, one sequence acts as the reference sequence with which test sequence is compared.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and subsequence coordinates are designated as needed, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence with reference sequence based on program parameters.

[0068] A preferred example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm described in Altschul et al. 1977 Nuc. Acids Res. 25:3389-3402 and Altschul et al. 1990 J. Mol. Biol. 215:403-410, respectively. BLAST software is publicly available on the World Wide Web at the National Center for Biotechnology Information (ncbi.nlm.nih.gov / ). Both default and other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3 and an expectation (E) of 10, and the BLOSUM62 scoring matrix alignment (B) of 50 (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)), an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0069] As used herein, the term "inhibit" refers to a measurable decrease in any biological activity. Thus, as used herein, "inhibit" or "inhibition" can be referred to as a percentage of normal levels of activity.

[0070] As used herein, the term "interleukin-15" or "IL15" refers to a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with a mammalian native IL15 amino acid sequence that is biologically active, i.e., the mutant protein ("mutein") has similar function (75% or greater) to the native IL15 protein in at least one functional assay. Functionally, IL15 is a cytokine that regulates the activation and proliferation of T cells and natural killer cells.

[0071] IL15 and IL2 share many biological activities, including binding to CD122, the IL2β / IL15β receptor subunit. The number of CD8+ memory cells is controlled by this balance between IL15 and IL2. IL15 induces activation of JAK kinases and phosphorylation and activation of the transcriptional activators STAT3, STAT5, and STAT6. IL15 also increases the expression of the apoptosis inhibitor BCL2L1 / BCL-X(L), possibly through its transcriptional activation activity of STAT6, thus preventing apoptosis. Two alternatively spliced ​​transcript variants of the IL15 gene encoding the same mature protein have been reported.

[0072] Exemplary functional assays for IL15 polypeptides include T cell proliferation (e.g., Montes et al. 2005 Clin Exp Immunol 142:292) and activation of NK cells, macrophages, and neutrophils. Methods for isolating specific immune cell subpopulations and detecting proliferation (i.e., H-thymidine incorporation) are well known in the art. Cell-mediated cytotoxicity assays can be used to measure the activation of NK cells, macrophages, and neutrophils. Cell-mediated cytotoxicity assays involving the release of isotopes (51Cr), dyes (e.g., tetrazolium, neutral red), or enzymes are also well known in the art, with commercially available kits (Oxford Biomedical Research, Oxford, M; Cambrex, Walkersville, MD; Invitrogen, Carlsbad, Calif.). IL15 has also been shown to inhibit Fas-mediated apoptosis (e.g., Demirci et al. 2004 Cell Mol Immunol 1:123). Apoptosis assays, including, for example, TUNEL and Annexin V assays, are well known in the art (e.g., Coliga et al. 1991-2006 Current Methods in Immunology John Wiley & Sons.), with commercially available kits (R&D Systems, Minneapolis, MN).

[0073] As used herein, the term "interleukin-15 receptor alpha" or "IL15Rα" refers to an interleukin-15 receptor alpha amino acid sequence derived from a mammal. Those skilled in the art will recognize that interleukin-15 receptor alpha nucleic acid and amino acid sequences are publicly available in genetic databases, such as GenBank via the World Wide Web at the National Center for Biotechnology Information (ncbi.nlm.nih.gov). Exemplary mammalian native IL-15 receptor alpha nucleic acid or amino acid sequences can be derived from, for example, humans, primates, dogs, cats, pigs, horses, cattle, sheep, rodents, mice, rats, hamsters, guinea pigs, etc. Accession numbers for exemplary mammalian native IL-15 nucleic acid sequences include NM_172200.1 (human isoform 2) and NM_002189.2 (human isoform 1 precursor). Accession numbers for exemplary native mammalian IL-15 amino acid sequences include NP_751950.1 (human isoform 2), and NP_002180.1 (human isoform 1 precursor).

[0074] As used herein, "interleukin-15 receptor alpha" or "IL15Rα" can also refer to a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a native mammalian IL15Rα amino acid sequence that is biologically active and has similar function (75% or greater) to the native IL15Rα protein in at least one functional assay. IL15Rα is a cytokine receptor that specifically binds IL15 with high affinity. One functional assay is specific binding to native IL15 protein.

[0075] As used herein, the term "isolated" refers to a molecule (such as a polypeptide or polynucleotide) that has been engineered to exist in greater concentrations than found in nature or that has been removed from its original environment. For example, a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, 20%, 40%, 50%, 70%, or 90% of the non-subject antibody material with which it is not naturally associated has been removed. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated." Furthermore, recombinant DNA molecules contained in a vector are considered isolated for purposes of the present invention. Isolated RNA molecules include the products of in vivo or in vitro RNA replication of DNA molecules and RNA molecules. Isolated nucleic acid molecules further include synthetically produced molecules. In addition, vector molecules contained in recombinant host cells are also isolated. Therefore, not all "isolated" molecules need to be "purified."

[0076] As used herein, the term "linker" or "linker segment" refers to a molecule or group that connects two other molecules or groups. A peptide linker can enable the connected molecules or groups to attain a functional configuration. The linker peptide preferably contains at least 2 amino acids, at least 3 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or approximately 100 amino acids.

[0077] The components of the fusion protein, such as the cytokine or other bioactive molecule and any peptide linker, can be organized in almost any manner, as long as the fusion protein has the intended function. In particular, each component of the fusion protein can be spaced from another component by at least one appropriate peptide linker segment or sequence, as needed. In addition, the fusion protein can include, for example, a tag to facilitate modification, identification, and / or purification of the fusion protein. More specific fusion proteins are described in the examples below.

[0078] As used herein, the term "low dose" refers to a dose that is at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) less than the minimum standard recommended dose of a particular compound formulated for a given route of administration for the treatment of any human disease or condition. For example, a low dose of a drug formulated for administration by inhalation will be different from a low dose of the same drug formulated for oral administration.

[0079] As used herein, the term "medium" or "media" includes any culture medium, solution, solid, semi-solid, or rigid support capable of supporting or containing bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and any host cells, including cellular contents. Thus, the term can encompass the medium in which a host cell has grown, e.g., the medium into which a polypeptide has been secreted, including the medium either before or after a growth step. The term can also encompass buffers or reagents containing host cell lysates, such as when a polypeptide is produced intracellularly and the host cells are lysed or disrupted to release the polypeptide.

[0080] As used herein, the term "modulate" refers to the direct or indirect production of an increase or decrease, stimulation, inhibition, interference, or blockage in a measured activity when compared to an appropriate control. A "modulator" of a polypeptide or polynucleotide refers to an entity that affects, e.g., increases, decreases, stimulates, inhibits, interferes, or blocks, the measured activity of the polypeptide or polynucleotide when compared to an appropriate control. For example, a "modulator" may bind to and / or activate or inhibit a target with measurable affinity, or may directly or indirectly affect the normal regulation of receptor activity.

[0081] The term "operably linked" refers to a functional linkage between a first nucleic acid sequence and a second nucleic acid sequence such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operatively linked nucleic acid sequences need not be physically contiguous to each other. The term "operably linked" also refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter or an array of transcription factor binding sites) and a transcribable nucleic acid sequence, where the expression control sequence directs transcription of the nucleic acid corresponding to the transcribable sequence.

[0082] As used herein, the term "pharmaceutically acceptable" excipient, carrier, or diluent refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting the pharmaceutical agent of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials that can serve as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and cellulose derivatives such as sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository wax; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition.

[0083] As used herein, the terms "polynucleotide," "nucleic acid molecule," "nucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to polymeric forms of nucleotides, including ribonucleotides and deoxyribonucleotides of any length. They can include both double-stranded, single-stranded, or triple-helical sequences, including, but not limited to, cDNA, mRNA, viral (e.g., DNA viruses and retroviruses) or genomic DNA sequences from prokaryotic sources, RNAi, cRNA, antisense molecules, recombinant polynucleotides, ribozymes, and synthetic DNA sequences. The term also encompasses sequences containing any of the known base analogs of DNA and RNA. Nucleotides can be referred to by their commonly accepted single-letter codes.

[0084] Polynucleotides are not limited to those polynucleotides as they occur in nature, but also include polynucleotides containing non-natural nucleotide analogs and internucleotide linkages. Nucleic acid molecules can contain modified nucleic acid molecules (e.g., modified bases, sugars, and / or nucleotide linkers). Non-limiting examples of this type of non-natural structure include polynucleotides in which the sugar is different from ribose, polynucleotides containing 3'-5' and 2'-5' phosphodiester linkages, and polynucleotides containing inverted linkages (3'-3' and 5'-5') and branched structures. Polynucleotides of the present invention also contain non-natural internucleotide linkages, such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), methyl phosphonate, phosphoramidate, C1-C6 alkyl phosphotriester, phosphorothioate, and phosphorodithioate-type C1-C4 alkyl phosphonate linkages. In all cases, polynucleotides of the present invention maintain the ability to hybridize to target nucleic acids in a manner similar to naturally occurring polynucleotides.

[0085] Unless otherwise indicated or apparent from the context, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly set forth sequence. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues. (Batzer et al. 1991 Nucleic Acid Res. 19:5081; Ohtsuka et al. 1985 J. Biol. Chem. 260:2605-2608; Rossolini et al. 1994 Mol. Cell. Probes 8:91-98.)

[0086] As used herein, the terms "prevent," "preventing," or "prevention" refer to a method for eliminating, delaying, avoiding, or halting the onset, occurrence, severity, or recurrence of a disease or condition. For example, a method is considered prophylactic if there is a reduction or delay in the onset, occurrence, severity, or recurrence of a disease or condition or one or more symptoms thereof in a subject susceptible to the disease or condition compared to a subject not receiving the method. The disclosed methods are also considered prophylactic if there is a reduction or delay in the onset, occurrence, severity, or recurrence of one or more symptoms of a disease or condition in a subject susceptible to the disease or condition after receiving the method compared to the subject's progression before receiving treatment. The reduction or delay in the onset, occurrence, severity, or recurrence of osteoporosis can be about a 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount therebetween.

[0087] Prevention etc. does not mean that the subject will never suffer from a particular disease or disorder.Prevention may require multiple administrations.Prevention may include the prevention of disease recurrence in subjects who have eliminated all disease symptoms, or the prevention of recurrence in relapsing-remitting disease.

[0088] As used herein, the term "promoter" refers to a DNA regulatory region capable of binding RNA polymerase in mammalian cells and initiating transcription of a downstream (3' direction) coding sequence operably linked to it. A promoter sequence contains the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels above background. A transcription initiation site and protein binding domains (consensus sequences) involved in RNA polymerase binding may be included within the promoter sequence. Eukaryotic promoters will often, but not always, contain "TATA" and "CAT" boxes. Promoters include those naturally adjacent to a nucleic acid molecule and those not naturally adjacent to a nucleic acid molecule. Furthermore, the term "promoter" encompasses inducible promoters, conditionally active promoters such as the cre-lox promoter, constitutive promoters, and tissue-specific promoters.

[0089] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like are included in this definition. Both full-length proteins and fragments thereof are included in the definition. These terms also include post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Furthermore, polypeptide may refer to proteins containing modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. These changes may be intentional or accidental. Amino acids may be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0090] As used herein, the term "purified" refers to a protein that is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., native cells, or, in the case of recombinantly produced proteins, host cells. Proteins that may be substantially free of cellular material include preparations that have less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating protein. When a protein or variant thereof is recombinantly produced by a host cell, the protein may be present at about 30%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When a protein or variant thereof is recombinantly produced by a host cell, the protein may be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of dry weight of cells. Thus, a "substantially purified" protein can have a purity level of at least about 80%, specifically at least about 85%, more specifically at least about 90%, at least about 95%, at least about 99%, and greater than 99%, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0091] Following their preparation, the proteins and prodrugs of the invention are preferably isolated and / or purified to obtain compositions containing 80% by weight or more ("substantially pure"), which are then used or formulated as described herein. In certain embodiments, the compounds of the invention are greater than 95% pure.

[0092] As used herein, the term "receptor" refers to a protein, including a glycoprotein or fragment thereof, that can interact with another molecule called a ligand. A ligand can belong to any class of biochemical or chemical compound. A ligand is typically an extracellular molecule that, upon binding to a receptor, typically initiates a cellular response, such as the initiation of a signal transduction pathway. A receptor is not necessarily a membrane-bound protein.

[0093] As used herein, the term "recombinant" with respect to a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin that, by reason of its origin or manipulation, is not linked to some or all of the polynucleotides with which it is naturally associated. The term "recombinant" with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

[0094] As used herein, the term "sample" refers to a sample from a human, animal, or research sample, such as a cell, tissue, organ, liquid, gas, aerosol, slurry, colloid, or coagulated material. A "sample" can be tested in vivo, for example, without removal from a human or animal, or it can be tested in vitro. A sample can be tested after processing, for example, by histological methods. A "sample" also refers to cells comprising, for example, a fluid or tissue sample, or cells separated from a fluid or tissue sample. A "sample" can also be a cell, tissue, organ, or liquid freshly taken from a human or animal, or a cell, tissue, organ, or liquid that has been processed or preserved.

[0095] As used herein, the term "soluble" refers to a fusion molecule, particularly a fusion protein, that does not readily sediment from an aqueous buffer, e.g., cell culture medium, under low G-force centrifugation (e.g., less than about 30,000 revolutions per minute in a standard centrifuge). A fusion molecule is soluble if it remains in aqueous solution at or near neutral pH, in the presence or absence of low concentrations of anionic or nonionic detergents, at temperatures above about 5-37°C. Under these conditions, the sedimentation value of a soluble protein is often low, e.g., less than about 10-50 Svedberg units.

[0096] Aqueous solutions referred to herein typically have a buffer compound to establish pH, ​​typically within a pH range of about 5 to 9, and an ionic strength range of about 2 mM to 500 mM. Protease inhibitors or mild non-ionic detergents may be added. Additionally, if desired, carrier proteins can be added (e.g., bovine serum albumin). Exemplary aqueous buffers include standard phosphate-buffered saline, Tris-buffered saline, or other well-known buffers and cell culture media formulations.

[0097] As used herein, the term "soluble IL15 receptor alpha" refers to a form of IL15 receptor alpha that lacks the transmembrane anchor portion of the receptor and therefore can be secreted from cells without being anchored to the plasma membrane.

[0098] As used herein, the term "stimulate" or "stimulating" refers to increasing, amplifying, enhancing, or boosting a physiological activity, such as an immune response. Stimulation can be a positive change. For example, the increase can be 5%, 10%, 25%, 50%, 75%, or even 90-100%. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0099] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to a living animal (human or non-human). A subject may be a mammal. The term "mammal" or "mammalian" refers to any animal within the taxonomic category Mammalia. A mammal may be a human or a non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term "subject" does not exclude an individual who is completely normal or normal in all respects with respect to a disease or condition.

[0100] As used herein, the term "inhibit" or "inhibiting" refers to decreasing, weakening, reducing, stopping, or stabilizing a physiological activity, e.g., an immune response. Inhibition can be a negative change. For example, the decrease can be 5%, 10%, 25%, 50%, 75%, or even 90-100%. Exemplary decreases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0101] As used herein, the term "therapeutically effective amount" refers to a dose of a therapeutic agent(s) sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be readily determined by one of ordinary skill in the art, for example, by administering a low dose of the agent initially and then gradually increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0102] As used herein, the term "transfected" means possessing introduced DNA or RNA, with or without the use of an accompanying facilitating agent such as lipofectamine. Methods of transfection known in the art include, for example, calcium phosphate transfection, DEAE-dextran transfection, protoplast fusion, electroporation, and lipofection.

[0103] As used herein, the term "treatment" or "treating" of a disease or disorder refers to a method of alleviating, delaying, or ameliorating such condition, or one or more symptoms of such disease or condition, before or after it occurs. Treatment can be directed at one or more effects or symptoms of the disease and / or underlying pathology. Treatment can be any alleviation, and can be, but is not limited to, complete ablation of the disease or disease symptoms. The degree of such reduction or prevention, as measured by standard techniques, is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100%, compared to an equivalent untreated control.

[0104] As used herein, the term "tumor" refers to any malignant or neoplastic cell.

[0105] As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring genetic material into a host cell or organism. Vectors are composed of either DNA or RNA. A vector has its own origin of replication, one or more unique recognition sites for restriction endonucleases that can be used to insert foreign DNA, and a conventional selectable marker such as a gene encoding antibiotic resistance, and often a recognition sequence (such as a promoter) for expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0106] Any composition or method disclosed herein can be combined with any one or more of the other compositions and methods provided herein. <Detailed Description of the Invention>

[0107] The present invention provides novel fusion proteins and therapeutic uses thereof. More specifically, the present invention provides novel fusion proteins of IL15 and its prodrugs, compositions thereof and methods for preparation thereof, which are useful for treating various diseases and disorders, such as hyperplasias, solid tumors or hematopoietic malignancies, with reduced off-target toxicity and side effects during treatment.

[0108] In one aspect, the present invention generally relates to a fusion protein comprising a first structural unit: a subunit of interleukin-15 receptor (IL15R) or a fragment thereof, a second structural unit: active IL15, a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein, and a first linker segment covalently linking the first, second, and third structural units, wherein the first structural unit is located at the N-terminus of the fusion protein and the second structural unit is located between the first and third structural units.

[0109] In another aspect, the present invention relates to a fusion protein as a whole, which comprises a first structural unit: a subunit of interleukin-15 receptor (IL15R) or a fragment thereof; a second structural unit: active IL15; a third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a linker segment covalently linking the first, second, and third structural units, wherein when the N-terminus of the fusion protein is the second structural unit, the first structural unit is located between the second structural unit and the third structural unit.

[0110] In certain embodiments of the fusion protein, the subunit of IL15R is selected from an α subunit, a β subunit, and a γ subunit.

[0111] In certain embodiments of the fusion protein, the subunit of IL15R is the alpha subunit.

[0112] In a particular embodiment of the fusion protein, the fragment is the sushi domain of the alpha subunit of mouse IL15R, having the amino acid sequence set forth in SEQ ID NO:4.

[0113] In a particular embodiment of the fusion protein, the fragment is the sushi domain of the alpha subunit of human IL15R, having the amino acid sequence set forth in SEQ ID NO:5.

[0114] In certain embodiments of the fusion protein, the IL15 is human or mouse IL15.

[0115] In certain embodiments of the fusion protein, the IL15 is murine IL15. In certain embodiments of the fusion protein, the murine IL15 has the amino acid sequence shown in SEQ ID NO:1.

[0116] In certain embodiments of the fusion protein, the IL15 is human IL15. In certain embodiments of the fusion protein, the human IL15 has the amino acid sequence shown in SEQ ID NO:2.

[0117] In certain embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc fragment.

[0118] In a particular embodiment of the fusion protein, the human Fc fragment comprises a human IgG1-Fc having the amino acid sequence set forth in SEQ ID NO:3.

[0119] In certain embodiments of the fusion protein, the linker segment L1 comprises multiple GGGS.

[0120] In a particular embodiment of the fusion protein, the linker segment L1 connecting the first structural unit to the third structural unit comprises the amino acid sequence shown in SEQ ID NO:9.

[0121] In a particular embodiment of the fusion protein, the linker segment L1 connecting the first and second structural units comprises the amino acid sequence shown in SEQ ID NO:8.

[0122] In a specific embodiment, the fusion protein further comprises a fourth structural unit located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor β subunit (Rβ); and a linker segment L2 covalently linking the fourth structural unit to the remaining structural units of the fusion protein, wherein the first structural unit is covalently linked to the C-terminus of the fourth structural unit, the second structural unit is located between the first structural unit and the third structural unit, and the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

[0123] In a specific embodiment, the fusion protein further comprises a fourth structural unit: the extracellular domain of the IL15 receptor β subunit (Rβ) located at the N-terminus of the fusion protein; and a linker segment L2 covalently linking the fourth structural unit to the remaining structural units of the fusion protein, wherein the second structural unit is covalently linked to the C-terminus of the fourth structural unit, the first structural unit is located between the second structural unit and the third structural unit, and the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in a tumor microenvironment.

[0124] In a specific embodiment, the amino acid sequence of mouse Rb has the amino acid sequence set forth in SEQ ID NO:6.

[0125] In a specific embodiment, the amino acid sequence of human Rb has the amino acid sequence set forth in SEQ ID NO:7.

[0126] In certain embodiments of the fusion protein, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

[0127] In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 9 (MMP9).

[0128] In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 14 (MMP14).

[0129] In certain embodiments of the fusion protein, the linker segment L2 comprises the amino acid sequence set forth in SEQ ID NOs: 10-23.

[0130] In yet another aspect, the present invention relates generally to homodimeric or heterodimeric proteins, including the fusion proteins disclosed herein.

[0131] In a specific embodiment, the homodimeric or heterodimeric protein comprises a monomer of RA-IL15-Fc: a fusion protein of human IgG1 Fc having the sushi domain of the mouse IL15 receptor α subunit, linker segment L1, mouse IL15, linker segment L1, e.g., the amino acid sequence set forth in SEQ ID NO: 24.

[0132] In a specific embodiment, the homodimeric or heterodimeric protein comprises a monomer of RA-IL15-Fc: a fusion protein of human IgG1 Fc having the sushi domain of the human IL15 receptor alpha subunit, linker segment L1, human IL15, linker segment L1, e.g., the amino acid sequence set forth in SEQ ID NO: 25.

[0133] In a specific embodiment, the homodimeric or heterodimeric protein comprises a fusion protein of a monomer of IL15-RA-Fc: mouse IL15, a linker segment L1, the sushi domain of the IL15 receptor α subunit, a linker segment L1, e.g., human IgG1 Fc having the amino acid sequence set forth in SEQ ID NO: 26.

[0134] In a specific embodiment, the homodimeric or heterodimeric protein comprises a fusion protein of IL15-RA-Fc monomer: human IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, e.g., human IgG1 Fc, having the amino acid sequence set forth in SEQ ID NO: 27.

[0135] In a specific embodiment, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: a fusion protein of human IgG1 Fc having the extracellular domain of mouse IL15 receptor β subunit, linker segment L2, mouse IL15, linker segment L1, the sushi domain of IL15 receptor α subunit, linker segment L1, e.g., the amino acid sequence set forth in SEQ ID NO: 28.

[0136] In a specific embodiment, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: a fusion protein of the extracellular domain of the human IL15 receptor β subunit, linker segment L2, human IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, and human IgG1 Fc having the amino acid sequence set forth in, for example, SEQ ID NOs: 29-41.

[0137] In a specific embodiment, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: a fusion protein of the extracellular domain of the human IL15 receptor β subunit, linker segment L1, human IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, e.g., human IgG1 Fc having the amino acid sequence set forth in SEQ ID NO: 42.

[0138] In certain embodiments, the homodimeric or heterodimeric protein is hydrolyzed by proteolytic enzymes that are specifically expressed in the tumor microenvironment.

[0139] In yet another aspect, the invention relates generally to substantially purified proteins, such as fusion proteins or fragments, disclosed herein.

[0140] In yet another aspect, the invention generally relates to polynucleotides encoding proteins, such as fusion proteins or fragments thereof, as disclosed herein.

[0141] In yet another aspect, the invention generally relates to an expression vector comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, as disclosed herein.

[0142] In yet another aspect, the invention generally relates to pharmaceutical compositions comprising a protein, such as a fusion protein or fragment thereof, disclosed herein and a pharmaceutically acceptable excipient, carrier, or diluent.

[0143] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent.

[0144] In yet another aspect, the present invention generally relates to a method for treating a disease or condition, the method comprising administering to a patient in need thereof a therapeutically effective amount of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof disclosed herein, wherein the disease or condition is selected from a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0145] In certain embodiments, the disease or condition being treated is hyperplasia.

[0146] In certain embodiments, the disease or condition being treated is a solid tumor.

[0147] In certain embodiments, the disease or condition being treated is a hematopoietic malignancy.

[0148] In certain embodiments, the subject being treated is further administered one or more of chemotherapy, radiation therapy, targeted therapy, immunotherapy, or hormone therapy.

[0149] In certain embodiments, the method further comprises administering a chemotherapeutic agent to the subject.

[0150] In certain embodiments, the method further comprises administering radiation therapy to the subject.

[0151] In certain embodiments, the method further comprises administering a targeted therapy to the subject.

[0152] In certain embodiments, the method further comprises administering immunotherapy to the subject.

[0153] In certain embodiments, the method further comprises administering hormone therapy to the subject.

[0154] As used herein, the term "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), Sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, GlaxoSmithKline), lonafamib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs), and gefitmib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU alkylating agents such as 5271; Sugen), thiotepa, and CYTOXAN® cyclophosphamide; alkylsulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic cousins ​​adozelesin, carzelesin, and bizelesin); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatin;Nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembine, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma 1 and calicheamicin omega 1 (Angew Chem. Intl. Ed. Engl. (1994) 33:183-186); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycin, actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, ethonib Mitomycins such as cin, idarubicin, marcelomycin, and mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine;Androgens such as calisthenol, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as aminoglutethimide, mitotane, and trilostane; folinic acid Folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil; amsacrine; bestravcil; bisantrene; edatrexate; defofamine; demecolcine; diaziconazole; elfornithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin, fenameth; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazine; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triazicon, 2,2',2''-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); clophosphamide; thiotepa; taxoids, such as TAXOL® (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (chromophore-free), albumin-engineered nanoparticle formulations of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111), and TAXOTERE® (docetaxel; Rhone-Poulenc Rorer, Antony, France); chlorambucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine);Novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0155] Examples of second (or additional) agents or therapeutic agents include immunotherapies (e.g., PD-1 inhibitors (pembrolizumab, nivolumab, cetuximab), PD-L1 inhibitors (atezolizumab, avelumab, durvalumab), CTLA4 antagonists, cell signaling inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, temsirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab, ranibizumab, pegaptanib, panitumumab, etc.), mitotic inhibitors (e.g., paclitaxel, Taxel, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, clomabucil, carmustine, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), intercalating anticancer agents (e.g., actinomycin, anthracyclines, bleomycin, mitomycin C, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide, etc.), immunotherapeutic agents (e.g., interleukins, interferons, etc.), and antihormonal agents (e.g., tamoxifen, raloxifene, etc.).

[0156] In yet another aspect, the invention relates generally to the use of a protein, such as a fusion protein or fragment thereof, disclosed herein to treat or alleviate a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0157] In yet another aspect, the invention relates generally to the use of polynucleotides encoding proteins such as the fusion proteins disclosed herein or fragments thereof to treat or alleviate a disease or disorder (e.g., hyperplasia, solid tumor, or hematopoietic malignancy).

[0158] In yet another aspect, the invention generally relates to the use of a protein, such as a fusion protein or fragment thereof disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0159] In yet another aspect, the invention generally relates to the use of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharmaceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0160] In certain embodiments, the agent is an anti-cancer agent.

[0161] In certain embodiments, the disease or disorder is one or more selected from head and neck cancer, endometrial cancer, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain cancer, stomach cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

[0162] In certain embodiments, the anti-cancer agent treats B-cell lymphoma or is effective against colon cancer.

[0163] In yet another aspect, the invention generally relates to cell lines comprising polynucleotides encoding proteins, such as fusion proteins or fragments thereof, as disclosed herein.

[0164] In yet another aspect, the invention generally relates to a method for producing a protein, comprising culturing a cell line, hi certain embodiments, the method further comprises purifying or isolating the produced protein, such as a fusion protein or fragment thereof, as disclosed herein.

[0165] In yet another aspect, the invention generally relates to a method for making a protein, the method comprising the steps of providing an expression vector encoding a protein, such as a fusion protein or fragment thereof disclosed herein, introducing the expression vector into a host cell, culturing the host cell in a medium under conditions sufficient to express the protein, and purifying the protein from the host cell or the medium.

[0166] Any suitable expression vector can be used. An exemplary expression vector is the pEE12.4 expression vector.

[0167] Any suitable host cell can be used, for example, 293F and CHO cells.

[0168] Introduction of the expression vector can be achieved by any suitable transfection method, and via transient transfection or stable cell lines.

[0169] Any suitable purification method can be used. Exemplary purification methods are by Protein A / G affinity chromatography or size exclusion.

[0170] In yet another aspect, the invention relates generally to isolated proteins produced by the methods disclosed herein.

[0171] In certain embodiments, an isolated protein is substantially pure.

[0172] As disclosed herein, linker sequences can be used to link two or more polypeptides of a biologically active polypeptide to generate a single-chain molecule having a desired functional activity.

[0173] Any suitable linker can be employed. Exemplary peptide linker sequences include those having about 7 to 20 amino acids, for example, about 8 to 16 amino acids. The linker sequence is preferably flexible so as not to hold the biologically active polypeptide or effector molecule in a single, undesired conformation. The linker sequence can be used, for example, to space the recognition site from the fused molecule. Specifically, the peptide linker sequence can be configured to provide molecular flexibility. The linker preferably contains primarily amino acids with small side chains, such as glycine, alanine, and serine, to provide flexibility.

[0174] Overall, preparation of the fusion protein complexes of the present invention can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques, including, for example, polymerase chain amplification reaction (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into appropriate cells, transformation or transfection of the host, and culture of the host. Additionally, fusion molecules can be isolated and purified using chaotropic agents and well-known electrophoretic, centrifugation, and chromatographic methods. (For disclosures related to these methods, see Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

[0175] The present invention further provides nucleic acid and DNA sequences encoding the fusion proteins described herein. The DNA sequences can be carried by vectors suitable for extrachromosomal replication, such as phages, viruses, plasmids, phagemids, cosmids, YACs, or episomes. For example, DNA vectors encoding the desired fusion proteins can be used to facilitate the preparation methods described herein and to obtain significant quantities of the fusion proteins or their components. The DNA sequences can be inserted into an appropriate expression vector, i.e., a vector containing the necessary elements for transcription and translation of the inserted protein-coding sequence. A variety of host-vector systems are available for expressing the protein-coding sequences. These may include mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with viruses (e.g., baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. Depending on the host-vector system utilized, any one of several appropriate transcription and translation elements can be used. (For disclosures relating to these methods, see Sambrook, el al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel, et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

[0176] The fusion protein components encoded by the DNA vector can be provided in a cassette format. The term "cassette" means that each component can be easily replaced with another component using standard recombinant techniques. In particular, DNA vectors configured in a cassette format are particularly desirable when the encoded fusion complex is to be used against pathogens that may express or possess serotypes.

[0177] To create a vector encoding a fusion protein complex, a sequence encoding a biologically active polypeptide is ligated to a sequence encoding an effector peptide using an appropriate ligase. DNA encoding the display peptide can be obtained by isolating DNA from natural sources, such as from a suitable cell line, or by known synthetic methods, such as the phosphotriester method (Oligonuclide OTIDE SYNTHESIS, IRL PRESS, MJGAIT, ED., 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. Once isolated, the gene encoding the biologically active polypeptide can be amplified by PCR or other means known in the art. Suitable PCR primers for amplifying the biologically active polypeptide gene may add restriction sites to the PCR product. The PCR product preferably contains splice sites for the effector peptide and leader sequence necessary for proper expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably contains a sequence encoding a linker sequence or a restriction enzyme site for ligating such a sequence.

[0178] The fusion proteins described herein can be produced by standard recombinant DNA techniques. For example, once a DNA molecule encoding a biologically active polypeptide is isolated, the sequence can be ligated to another DNA molecule encoding an effector polypeptide. The nucleotide sequence encoding the biologically active polypeptide can be directly linked to the DNA sequence encoding the effector peptide, or more typically, a DNA sequence encoding a linker sequence as discussed herein can be inserted between the sequences encoding the biologically active polypeptide and the effector peptide and ligated using an appropriate ligase. The resulting hybrid DNA molecule can be expressed in a suitable host cell to produce a fusion protein complex. The DNA molecules are ligated to each other in a 5'-3' orientation (i.e., the DNA molecules are ligated to each other in-frame) so that the translation frame of the encoded polypeptide is not altered after the ligation reaction. The resulting DNA molecule encodes an in-frame fusion protein.

[0179] Other nucleotide sequences can also be included in the genetic construct. For example, a promoter sequence controlling expression of a sequence encoding a biologically active polypeptide fused to an effector peptide, or a leader sequence that directs the fusion protein to the cell surface or culture medium, can be included in or present in the expression vector into which the construct is inserted.

[0180] In obtaining a mutant biologically active polypeptide, IL15, IL15R, or Fc domain coding sequence, one skilled in the art will recognize that the polypeptide can be modified by specific amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, it is well known that conservative amino acid substitutions, i.e., replacing one amino acid with another of similar size, charge, polarity, and conformation, are unlikely to significantly alter the function of the protein. The 20 standard amino acids that are the building blocks of proteins can be roughly categorized into four groups of conserved amino acids: the nonpolar (hydrophobic) group includes alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; the polar (uncharged, neutral) group includes asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine; the positively charged (basic) group includes arginine, histidine, and lysine; and the negatively charged (acidic) group includes aspartic acid and glutamic acid. Substitution of one amino acid in a protein with another within the same group is unlikely to adversely affect the protein's biological activity. In other instances, modifications to amino acid positions can be made to reduce or enhance a protein's biological activity. Such changes can be introduced randomly or via site-directed mutagenesis based on the known or predicted structural or functional properties of the target residue. Following expression of the mutant protein, changes in biological activity due to the modification can be readily assessed using binding or functional assays.

[0181] Homology between nucleotide sequences can be determined by DNA hybridization analysis, in which the stability of double-stranded DNA hybrids depends on the degree of base pairing present. High temperature and / or low salt conditions can be varied to reduce hybrid stability and prevent annealing of sequences with less than a selected degree of homology. For example, for a sequence with a GC content of approximately 55%, hybridization and washing conditions of 40-50°C, 6x SSC (sodium chloride / sodium citrate buffer), and 0.1% SDS (sodium dodecyl sulfate) indicate approximately 60-70% homology; hybridization and washing conditions of 50-65°C, 1x SSC, and 0.1% SDS indicate approximately 82-97% homology; and hybridization and washing conditions of 52°C, 0.1x SSC, and 0.1% SDS indicate approximately 99-100% homology. A wide range of computer programs are also available for comparing nucleotide and amino acid sequences (and measuring the degree of homology). Readily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW programs, respectively.

[0182] Many strategies can be used to express the protein fusion complexes of the present invention. For example, the fusion protein construct can be incorporated into a suitable vector by known methods using restriction enzymes to create an opening in the vector for insertion of the construct, followed by subsequent ligation. The vector containing the gene construct is then introduced into a suitable host for expression of the fusion protein. (Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989) for disclosure relating to these methods.)

[0183] Selection of an appropriate vector can be performed empirically based on factors related to the cloning protocol. For example, the vector must have an appropriate response control for the host being used. Furthermore, the vector must be capable of accommodating the DNA sequence encoding the fusion protein complex to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those that are easily transformed and capable of rapid growth in culture medium. Specifically, preferred host cells include prokaryotes, such as E. coli and Bacillus subtillus, and eukaryotes, such as animal cells and yeast strains, e.g., S. cerevisiae. Mammalian cells are generally preferred, particularly J558, NSO, SP2-O, or CHO. Other suitable hosts include insect cells, such as Sf9. Conventional culture conditions can be employed. See Sambrook, supra. Stably transformed or transfected cell lines can be selected. Cells expressing the fusion protein complexes of the present invention can be determined by known procedures. For example, expression of the fusion protein complex bound to the immunoglobulin can be determined by ELISA and / or immunoblotting specific for the bound immunoglobulin. Other methods for detecting expression of fusion proteins containing a biologically active polypeptide bound to an IL12 or IL12R domain are disclosed in the Examples.

[0184] Host cells can be used for preparative purposes to propagate nucleic acids encoding the desired fusion protein or its components. Host cells can include prokaryotic or eukaryotic cells specifically intended for production of the fusion protein. Host cells therefore include, inter alia, yeast, fly, worm, plant, frog, mammalian cells, and organisms capable of propagating nucleic acids encoding the fusion. Non-limiting examples of mammalian cell lines that can be used include CHO dhfr cells (Urlaub and Chasm, 1980 Proc. Natl. Acad. Sci. USA, 77:4216), 293 cells (Graham et al., 1977 J. Gen. Virol., 36:59()), or myeloma cells such as SP2 or NSO (Galfre and Milstein, 1981 Meth. Enzymol, 73(B):3).

[0185] Host cells into which nucleic acids encoding the desired fusion protein complexes can be propagated include insects (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha, generally reviewed by Fleer, R., 1992 Current Opinion in Biotechnology, 3(5):486-496), as well as non-mammalian eukaryotic cells. Certain prokaryotes, such as E. coli and Bacillus, are also contemplated.

[0186] Nucleic acids encoding the desired fusion proteins can be introduced into host cells by standard techniques for transfecting cells. The terms "transfecting" or "transfection" are intended to encompass all conventional techniques for introducing nucleic acids into host cells, including calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, viral transduction and / or integration.

[0187] Various promoters (transcription initiation regulatory regions) can be used in accordance with the present invention. The selection of an appropriate promoter depends on the proposed expression host. Promoters from heterologous sources can be used as long as they function in the selected host.

[0188] The choice of promoter depends on the desired efficiency and level of peptide or protein production. Inducible promoters, such as tac, are often employed to dramatically increase the level of protein expression in E. coli. Protein overexpression can be harmful to the host cell, resulting in limited growth of the host cell. Using an inducible promoter system allows host cells to be cultured to an acceptable density before inducing gene expression, facilitating higher product yields.

[0189] A variety of signal sequences may be used in accordance with the present invention. Signal sequences that are homologous to the biologically active polypeptide coding sequence may be used. Alternatively, a signal sequence selected or designed for efficient secretion and processing in the expression host may be used. The signal sequence may be directly linked to the protein coding sequence through a sequence encoding a signal peptidase cleavage site, or may be linked via a short nucleotide bridge.

[0190] Expression constructs can be assembled using known recombinant DNA techniques. Restriction enzyme digestion and ligation are the basic steps used to join two fragments of DNA. Polylinkers and adapters can be used to facilitate the joining of selected fragments. Expression constructs can typically be assembled in stages using rounds of restriction, ligation, and transformation of E. coli. Numerous cloning vectors suitable for expression construction are known in the art (e.g., λ1ZAR and pBLUESCRIPT SK-1, Stratagene, La Jolla, Calif.; pET, Novagen Inc., Madison, Wis.; rEE12.4, Lonza Bioologies, Basel, Switzerland).

[0191] The expression construct may be transformed into a host cell as a cloning vector construct, or may be used in a linear or circular form, or removed from the cloning vector, or introduced into a delivery vector. This delivery vector facilitates the introduction and maintenance of the expression construct in the selected host cell type. The expression construct is introduced into the host cell by any of the known gene transfer systems (e.g., natural competence, chemically mediated transformation, protoplast transformation, electroporation, biolistic transformation, transfection, or conjugation). The gene transfer system selected depends on the host cell and vector system used.

[0192] The present invention further provides a manufacturing process for isolating a desired fusion protein. In this process, a nucleic acid encoding the desired protein operably linked to a regulatory sequence is introduced into host cells (e.g., yeast, fungal, insect, bacterial, or animal cells) and grown at a production scale in culture to stimulate transcription of the nucleotide sequence encoding the desired fusion protein. The desired fusion protein is then isolated from harvested host cells or from the culture medium. Standard protein purification techniques can be used to isolate the protein of interest from the medium or harvested cells. In particular, purification techniques can be used to express and purify the desired fusion protein on a large scale (i.e., at least milligram quantities) from a variety of platforms, including roller bottles, spinner flasks, tissue culture plates, bioreactors, or fermenters.

[0193] The expressed protein fusion complex can be isolated and purified by known methods. Typically, the culture medium is centrifuged or filtered, and the supernatant is then purified by affinity or immunoaffinity chromatography, for example, protein A or protein G affinity chromatography or immunoaffinity protocols include the use of monoclonal antibodies that bind the expressed fusion complex, such as the linked TCR or immunoglobulin region. The fusion proteins of the present invention can be isolated and purified by an appropriate combination of known techniques. These methods include, for example, methods that utilize solubility such as salt precipitation and solvent precipitation; methods that utilize differences in molecular weight such as dialysis; ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that utilize differences in charge such as ion exchange column chromatography; methods that utilize specific affinity such as affinity chromatography; methods that utilize differences in hydrophobicity such as reversed-phase high performance liquid chromatography; and methods that utilize differences in isoelectric point such as isoelectric focusing (Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989) for disclosure relating to these methods).

[0194] The fusion proteins of the present invention are preferably substantially pure, i.e., separated from the cellular components that naturally accompany them, so that the fusion protein is present in at least 80% or 90%-95% homogeneity (w / w). Fusion proteins with at least 98-99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical, and research applications. Once substantially purified, the fusion protein should be substantially free of contaminants for therapeutic use. Once partially or substantially purified, the soluble fusion protein can be used therapeutically or in in vitro or in vivo assays as disclosed herein. Substantial purity can be determined by a variety of standard techniques, such as chromatography and gel electrophoresis.

[0195] The present invention also provides a pharmaceutical formulation comprising a therapeutically effective amount of a composition, fusion protein, polynucleotide, genetic construct, vector or host cell according to the invention and a pharmaceutically acceptable excipient or vehicle.

[0196] Preferred excipients for use in the present invention include sugars, starches, celluloses, gums, and proteins. In a preferred embodiment, the pharmaceutical compositions of the present invention are formulated in pharmaceutical forms for administration as solids (e.g., tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide liquid forms, etc.), liquids (e.g., solutions, suspensions, emulsions, elixirs, lotions, astringents, etc.), or semisolids (gels, ointments, creams, and the like). The pharmaceutical compositions of the present invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intravenous, ventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, subcutaneous, or rectal. A revised version of the principles of administration for different forms of administration, excipients used, and procedures for their preparation can be found in Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000) Examples of pharmaceutically acceptable solvents are known in the state of the art and include saline solutions buffered with phosphates, water, emulsions such as emulsions, different types of humectants, sterile solutions, etc. Compositions containing said solvents can be formulated by conventional procedures known in the state of the art.

[0197] In addition, in the case of pharmaceutical compositions of the invention comprising a nucleic acid (a polynucleotide, vector or gene construct of the invention), the invention contemplates pharmaceutical compositions specifically formulated for administering said nucleic acid, which have the advantage of administering said nucleic acid in its naked form, i.e., of compounds that protect the nucleic acid from degradation by the organism's nucleases, eliminating the toxicity associated with the reagents used for transfection. Suitable routes of administration for naked compounds include intravascular, intratumoral, intracranial, intraperitoneal, splenic, intramuscular, subcutaneous, mucosal, topical, and oral routes (Templeton, 2002 DNA Cell Biol., 21:857-867). Alternatively, nucleic acids can be administered in liposome-forming moieties, conjugated to cholesterol, or conjugated to compounds capable of facilitating translocation across cell membranes, such as the TAT peptide derived from the HIV-1 TAT protein, the third helix of the homeodomain of the D. melanogaster antennapedia protein, the VP22 protein of herpes simplex virus, oligomers of arginine, and peptides such as those described in WO07069090 (Lindgren, et al. 2000 Trends Pharmacol Sci 21:99-103; Schwarze, et al. 2000 Trends Pharmacol Sci 21:45-48; Lundberg, et al. 2003 Mol. Therapy 8:143-150; and Snyder, et al. 2004 Pharm. Res. 21:389-393). Alternatively, the polynucleotide can be administered in a moiety that forms part of a plasmidine vector or viral vector, preferably an adenovirus-based vector, an adeno-associated virus, or a retrovirus, such as a virus based on murine leukemia virus (MLV) or a lentivirus (HIV, FIV, EIAV).

[0198] The compositions of the present invention can be administered at a dose of less than 10 mg per kilogram of body weight, preferably less than 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001 mg per kg of body weight, and less than 200 nmol of drug, in other words, less than about 4.4 x 10 copies per kg of body weight or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, or 0.075 nmol per kg of body weight. A single dose can be administered by injection, inhalation, or topical administration. The bifunctional polynucleotides and compositions of the invention can be administered directly to the organ in which the target mRNA is expressed, in which case the dose is between 0.00001 mg and 3 mg per organ, or preferably between 0.0001 and 0.001 mg per organ, between about 0.03 and 3.0 mg per organ, between about 0.1 and 3.0 mg per organ, or between 0.3 and 3.0 mg per organ.

[0199] The dosage depends on the severity and response to the condition being treated and may vary from several days to several months, or until the condition recurs. The optimal dosage can be determined by periodically measuring the concentration of the drug in the patient's organism. The optimal dosage can be determined from the EC50 value obtained through previous in vitro or in vivo testing in animal models. This unit dosage can be administered once daily or less than once daily, preferably less than once every 2, 4, 8, or 30 days. Alternatively, an initial dose can be administered, followed by one or more maintenance doses, which are generally smaller than the initial dose. A maintenance regimen may involve treating the patient with a dose ranging from 0.01 μg to 1.4 mg per kg of body weight per day, for example, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of body weight per day. The maintenance dose is preferably administered no more than once every 5, 10, or 30 days. Treatment must be continued for a period of time that varies depending on the type of changes the patient experiences, their severity, and the patient's condition. After treatment, the patient's evolution must be monitored to determine whether the dose should be increased in the event of disease that does not respond to treatment, or whether the dose should be decreased if improvement of the disease or undesirable secondary effects are observed.

[0200] The daily dose can be administered as a single dose or two or more doses, depending on the specific situation. If repeated or frequent administration is required, implantation of an administration device such as a pump, semi-permanent catheter (intravenous, intraperitoneal, intraluminal, or intracapsular) or reservoir is recommended.

[0201] The compositions of the present invention may be administered in accordance with methods known to those skilled in the art, including, but not limited to, intravenous, oral, nasal, parenteral, topical, transdermal, rectal, and the like.

[0202] The following examples are meant to illustrate the practice of the present invention and are not meant to limit it in any way. [Example]

[0203] The following examples illustrate certain exemplary embodiments of compounds prepared in accordance with the disclosed invention. It is understood that the following general methods, and other methods known to those skilled in the art, are applicable to compounds and subclasses and species as disclosed herein.

[0204] Example 1. Construction of fusion proteins A. Construction of four fusion proteins Control protein construct: IL15-Fc Mouse IL15 was fused to the N-terminus of hIgG Fc (designated IL15-Fc) as shown in Figure 1A. The amino acid sequence of mouse IL15 was SEQ ID NO: 1. The amino acid sequence of hIgG Fc was SEQ ID NO: 3.

[0205] Two formats of Super IL15 Schematic diagrams of IL15Rα sushi-IL15-Fc (designated RA-IL15-Fc) and IL15-IL15Rα sushi-Fc (designated IL15-RA-Fc) are shown in Figures 1B and 1C. Due to similar biological activity, both structures are interchangeably referred to as super-IL15. The amino acid sequence of mouse IL15Rα sushi is SEQ ID NO: 4. The amino acid sequence of human IL15Rα sushi is SEQ ID NO: 5. The sequence of the linker between IL15Rα sushi and IL15 is SEQ ID NO: 8. Mouse RA-IL15-Fc and IL15-RA-Fc are fully represented by SEQ ID NO: 24 and SEQ ID NO: 26. Human RA-IL15-Fc and IL15-RA-Fc are fully represented by SEQ ID NO: 25 and SEQ ID NO: 27.

[0206] Prodrug The ECD (extracellular domain) of IL15Rβ was fused to the N-terminus of IL15-RA-Fc and connected by the linker segment L2.

[0207] A schematic diagram of IL15RβECD-L2-IL15-IL15Rα sushi-Fc (designated RB-IL15-RA-Fc) is shown in Figure 1D. The amino acid sequence of mouse IL15RβECD is SEQ ID NO: 6. The amino acid sequence of human IL15RβECD is SEQ ID NO: 7. Linker segment L2 is a substrate for MMP9 or MMP14. The amino acid sequence of the MMP9 substrate linker is SEQ ID NO: 10. The amino acid sequences of the MMP14 substrate linker are SEQ ID NOs: 11-23.

[0208] B. Fusion Protein Construction, Transfection, Expression, and Purification The gene was cloned into an expression vector such as pEE12.4. The plasmid was transiently transfected into 293F cells. Supernatants were collected 4-7 days after transfection. The fusion proteins were purified using protein A-Sepharose. Total proteins were quantified by ELISA and SDS-PAGE.

[0209] The detailed protocol is as follows:

[0210] Fusion protein construction The IL15, IL15RA, and IL15RB ECDs were synthesized and cloned into the pEE12.4-IgGκ-hIgG1 Fc plasmid, which contains a mouse IgGκ lead sequence and a human IgG1 Fc sequence. Plasmids were extracted using a standard commercially available plasmid extraction kit and stored at -80°C.

[0211] Fusion protein transfection 293F cells were cultured in CD OptiCHO™ medium and incubated in a 37°C, 8% CO2 incubator with shaking at 135 rpm. Cells were plated at a density of 0.6-0.8 x 106 cells / mL two days before transfection. Cells were harvested at a density of approximately 2.5-3.5 x 106 cells / mL, washed with Freestyle 293 medium, and resuspended in 200 mL of Freestyle 293. DNA (600 μg) was diluted in 5 mL of Freestyle 293 and filtered through a 0.22 μm filter. PEI (1.8 mg) was diluted in 5 mL of Freestyle 293 and filtered through a 0.22 μm filter. The DNA and PEI were mixed, incubated at room temperature for 5 minutes, and mixed with the cells in the flask. The flask was placed in a 37°C, 8% CO2 incubator with shaking at 85 rpm. 200 mL of EX-CEFF™ 293 medium was added 4 hours after transfection at 135 rpm. 20 hours after transfection, 3.8 mM VPA was added. Supernatants were harvested 4 to 7 days after transfection while cell viability was greater than 70%.

[0212] Purification of fusion proteins The fusion protein was purified using a protein A-Sepharose column according to the manual (Repligen Corporation). Binding buffer: 20 mM sodium phosphate, pH 7.0 Elution buffer: 0.1M glycine, pH 2.7 Regeneration buffer: 1M NaOH Neutralization buffer: 1M Tris-HCl, pH9.0 All buffers were filtered through a 0.45 μm filter. (1) Samples were centrifuged at 8000× rpm for 2 hours to remove cells and then filtered through a 0.45 μm filter. NaN3 was added to a final concentration of 0.05% to prevent bacterial growth. (2) When the column was stored with 20% ethanol, it was washed with 5 column volumes of distilled water at a linear flow rate of 50 to 100 cm / h. (3) The column was washed with 5-10 column volumes of elution buffer to wash away impurities. (4) The column was equilibrated with 5-10 column volumes of binding buffer at a linear flow rate of 50 to 100 cm / hr. (5) The pretreated sample was applied to the column. (6) The column was washed with 5-10 column volumes of binding buffer. (7) The column was eluted into a 1.5 mL collection tube.

[0213] The results of SDS-PAGE electrophoresis of the purified fusion proteins are shown in Figure 2 , where lane S1 was loaded with IL15-Fc, lane S2 was loaded with super-IL15, and lane S3 was loaded with RB-IL15-RA-Fc.

[0214] Example 2: Biological function of super-IL15 fusion protein A. Function of promoting lymphocyte proliferation Interleukin 15 (IL-15) was initially characterized by its ability to stimulate the proliferation of the murine T cell line CTLL-2. In this protocol, CTLL-2 cells are cultured in the presence of serial dilutions of murine IL-15, and their proliferation is measured by CCK8.

[0215] The following procedure was used: (1) Use CTLL-2 assay medium supplemented with 100 U / mL recombinant human IL-2 to culture CTLL2 cells. (2) CTLL-2 cells are harvested in logarithmic growth phase 24-48 hours after passage and washed twice to remove residual IL-2. Resuspend the cells in 5-10 mL CTLL-2 assay medium, count the cells, and adjust the concentration to 2 x 10 cells / mL. (3) Dilute the sample using CTLL-2 assay medium. The initial top concentration is 10 μg / mL. Serial dilutions of 1:10 are performed in seven tubes. (4) Add 100 μL of cell suspension to each flat-bottom 96-well plate (2 × 10 cells / well). Add 100 μL of sample to each well. Include a row of wells containing 200 μL assay medium only as a negative control. (5) Cover the plate and incubate for 48-72 hours. (6) Add 20 μL of CCK8. After 2-4 hours, read the OD450 and OD630 of each well using a microtiter plate reader.

[0216] The results are shown in Figure 3, which demonstrates that (1) the biological activities of the two forms of super-IL15 were similar, i.e., in the fusion protein, the function of super-IL15 was not affected regardless of whether it was the IL15 fragment or the IL15α sushi fragment, and (2) super-IL15 had approximately 100-fold increased biological activity compared to IL15-Fc.

[0217] B. Fusion fragment of IL15RβECD can block the biological function of super-IL15 The proliferative abilities of murine RB-IL15-RA-Fc and super-IL15 against CTLL2 were examined by CCK8 assay. The results, shown in Figure 4, indicate that the biological activity of RB-IL15-RA-Fc was reduced 100-fold, indicating that the extracellular domain of IL-15Rβ can block the biological function of super-IL-15.

[0218] C. Antitumor Efficacy and Systemic Toxicity in Different Tumor Models A20 model Experiment 1 (25 μg): A20 cells (3 × 106) were injected subcutaneously into the right flank of Balb / c mice. Tumor-bearing mice (60-80 mm3) were treated intratumorally (it) and intravenously (iv) with 25 μg of Super-IL15 on days 10 and 13. The control group was treated with PBS. Tumor volume = length × width × height / 2. Tumor growth curves were recorded.

[0219] Results: (1) In the intratumoral treatment group, tumors from all mice showed complete regression (Figure 5A). Mice that had already undergone complete tumor regression were re-challenged with a lethal dose of A20 cells. All mice rejected the re-challenged tumors and demonstrated a strong memory response (Figure 5C). (2) All mice died after the second intravenous treatment, indicating severe systemic toxicity (Figure 5B).

[0220] Experiment 2 (12.5 μg): A20 cells (3 × 10) were injected subcutaneously into the right flank of Balb / c mice. Tumor-bearing mice (60-80 mm) were treated intratumorally (it) and intravenously (iv) with 12.5 μg of Super-IL15 on days 10 and 13. The control group was treated with PBS. Tumor volume was defined as length × width × height / 2. Tumor growth curves were recorded.

[0221] Results (Figure 7): In the intratumorally administered treatment group, 100% of the mice had complete regression. In contrast, 20% of the intravenously treated mice had complete regression, and the remaining mice's tumors were partially suppressed.

[0222] MC38 model Experiment: MC38 cells (5 × 10 ) were injected subcutaneously into the right flank of C57 mice. Tumor-bearing mice (60 mm ) were treated intravenously with 25 μg of Super IL15 intratumorally (it) and intravenously (iv) on days 7 and 10. The control group was treated with PBS. Tumor volume = length × width × height / 2. Tumor growth curves were recorded.

[0223] Results: In the intratumorally administered treatment group, 50% of mice had complete regression (Figure 6A), and survival was significantly increased (Figure 6B). In contrast, mice administered intravenously did not have complete tumor regression (Figure 6A), and survival was slightly increased (Figure 6B).

[0224] The above results indicate that super-IL15 appears to function locally in the tumor microenvironment (TME).

[0225] Example 3: Comparison of tumor therapeutic effects and side effects between super IL15 and RB-IL15-RA-Fc A20 cells (3 × 106) were injected subcutaneously into the right flank of Balb / c mice. Tumor-bearing mice (60-80 mm3) were treated intraperitoneally (ip) with 12.5 μg of super-IL15 or Rβ-IL15-RA-Fc on days 10 and 13. Tumor growth was measured twice weekly. Serum was collected 20 hours after the second injection. Serum cytokine concentrations were measured by Cytometric Bead Array (CBA), and tumor curves were recorded.

[0226] The following CBA protocol was used: (1) Serum is collected from the ophthalmic vein and stored at -80°C. (2) Evaluate IL12p70, IL-6, IFN-γ, TNFα, MCP1, and IL-10 in serum using CBA kits from BD. (3) The standards were reconstituted in 2.0 mL of assay diluent and then recalibrated at room temperature for at least 15 minutes. The standards were serially diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256. (4) The Th1 / Th2 / Th17 cytokine capture beads were mixed. The number of assay tubes (including standards and controls) required for the experiment was determined. Each capture bead suspension was vortexed vigorously for 3-5 seconds before mixing. A 2 μL aliquot of each capture bead was added to one tube for each assay tube to be analyzed. A 10 μL aliquot of mouse Th1 / Th2 / Th17 PE detection reagent was added to the tube and vortexed thoroughly. (5) Th1 / Th2 / Th17 cytokine assay was performed: The mixed capture beads were vortexed and 20 μL was added to all assay tubes. 50 μL of mouse Th1 / Th2 / Th17 cytokine standard dilutions were added to control tubes. 50 μL of each unknown sample was added to the appropriately labeled sample assay tubes. The assay tubes were incubated at room temperature for 2 hours, protected from light. (6) 1 mL of wash buffer was added to each assay tube and centrifuged at 300 g for 5 minutes. (7) The supernatant was carefully aspirated and discarded from each assay tube. 300 μL of wash buffer was added to each assay tube to resuspend the bead pellet. (8) Samples were analyzed via flow cytometry, and cytokine levels were calculated according to standards.

[0227] This therapeutic effect is shown in Figure 8A, which demonstrates that the therapeutic effect of intravenously administered RB-IL15-RA-Fc is similar to that of super-IL15.

[0228] The results of the comparison of serum inflammatory factor levels are shown in Figure 8B and demonstrate that the toxic side effects of RB-IL15-RA-Fc are significantly reduced compared to super-IL15.

[0229] A20 cells (3 × 106) were injected subcutaneously into the right flank of Balb / c mice. Tumor-bearing mice (60-80 mm3) were treated intraperitoneally (ip) with 25 μg of super-IL15 or RB-IL15-RA-Fc on days 10 and 13. Tumor growth was measured twice weekly. Serum was collected 20 hours after the second injection. Serum cytokine levels were measured by Cytometric Bead Array (CBA), and tumor curves were recorded.

[0230] Results: After tumor rechallenge and treatment with super-IL15, tumor-bearing mice became significantly sicker, with severe weight loss, decreased mobility, and wrinkled fur, and all died within 1 day of the second treatment. In contrast, none of the mice treated with RB-IL15-RA-Fc died, and none appeared unhealthy. The survival curve for RB-IL15-RA-Fc was significantly longer than that for super-IL15. The survival curves are shown in Figure 9A, and serum inflammatory factor levels are shown in Figure 9B.

[0231] In summary, RB-IL15-RA-Fc reduced the toxic side effects of super-IL15.

[0232] Similar human versions of various IL15 fusion proteins and prodrugs have also been generated and tested in vitro. Production of the human proteins followed the cloning, transfection, and purification protocols previously described for production of the murine proteins.

[0233] Recombinant human MMP-14 / MT1-MMP (R&D Systems) was activated and incubated with the IL15 fusion protein at 37°C for 24 hours to confirm prodrug activation and cleavage at the L2 linker site.

[0234] SDS-PAGE electrophoresis results of purified human fusion protein incubated with or without MMP14 at 37° C. for 24 hours are shown in FIG.

[0235] The function of human RB-IL15-RA-Fc was measured using the HEK-Blue™ IL2 reporter cell assay (Invivogen). Upon IL-2 stimulation, HEK-Blue™ IL-2 cells induce STAT5 activation and secretion of a subsequence of SEAP. The level of STAT5-induced SEAP can be easily monitored using QUANTI-Blue™. Because IL15 binds and signals through a complex consisting of the IL-2 / IL-15 receptor β chain and the common γ chain, the HEK-Blue™ IL-2 cell line can also be used to measure IL15 and / or pro-IL15 functional activity.

[0236] The following HEK-Blue IL-2 reporter assay was used. (1) HEK-Blue IL-2 cells were gently rinsed in PBS and suspended in fresh, pre-warmed test medium (DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated PBS (56°C, ~1 x 106 cells / mL for 30 min). (2) Serially dilute the samples in a flat-bottom 96-well plate and incubate with 50 μL of cell suspension (~50,000 cells) per well in a CO2 incubator at 37°C for 20-24 hours. (3) 20 μL of induced HEK-BLUE IL-2 cell supernatant per well of a flat-bottom 96-well plate is incubated with 100 μL of resuspended QUANTI-Blue™ solution per well in a 37° C. incubator for 15 minutes to 1 hour. (4) determining SEAP levels using a spectrophotometer at 650 nm;

[0237] Results: Figure 11 demonstrates that RB-L2-15RA-Fc, constructed with an MMP14 substrate sequence embedded in the linker segment (L2) and incubated with MMP14, exhibited the same level of function as 15RA-Fc, both with and without MMP14. Consistently, RB-L1-15RA-Fc, constructed without the MMP14 substrate sequence embedded in the linker segment (L1), performed similarly to the sample without MMP14. The construct designation 15RA is an abbreviation for IL15-L1-RA. <array list> SEQ ID NO: 1: Mouse IL15 NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS SEQ ID NO: 2: Human IL15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO: 3: Human IgG1-Fc EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4: Mouse Rα-sushi domain GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT SEQ ID NO: 5: Human Rα-sushi domain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO: 6: Mouse Rβ extracellular domain AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINNVVCWEEKGWRRVKTCDFHPFDNLRL VAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE SEQ ID NO: 7: Human Rβ extracellular domain AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT SEQ ID NO: 8: Linker segment L1 SGGGSGGGGSGGGGSGGGGSGGGSLQ SEQ ID NO: 9: Linker segment L1 GGGGS SEQ ID NO: 10: Linker segment L2 (MMP9)GGGGSPVGLIGGGGGS SEQ ID NO: 11: Linker segment L2 (MMP14) GGGGSSGARYRWLTAGGGGS SEQ ID NO: 12: Linker segment L2 (MMP14) GGGGSSGRIGFLRTAGGGGS SEQ ID NO: 13: Linker segment L2 (MMP14) GGGGSSGAIGFLRTAGGGGS SEQ ID NO: 14: Linker segment L2 (MMP14) GGGGSSGRAMHMYTAGGGGS SEQ ID NO: 15: Linker segment L2 (MMP14) GGGGSSGAAMHMYTAGGGGS SEQ ID NO: 16: Linker segment L2 (MMP14) GGGGSSGRSENIRTAGGGGS SEQ ID NO: 17: Linker segment L2 (MMP14) GGGGSSGASENIRTAGGGGS SEQ ID NO: 18: Linker segment L2 (MMP14) GGGGSSGRPENIRTAGGGGS SEQ ID NO: 19: Linker segment L2 (MMP14) GGGGSSGAPENIRTAGGGGS SEQ ID NO: 20: Linker segment L2 (MMP14) GGGGSSGLISHSITAGGGGS SEQ ID NO: 21: Linker segment L2 (MMP14) GGGGSSGNLRSKLTAGGGGS SEQ ID NO: 22: Linker segment L2 (MMP14) GGGGSSGVFSIPLTAGGGGS SEQ ID NO: 23: Linker segment L2 (MMP14) GGGGSSGIKYHSLTAGGGGS SEQ ID NO: 24: Mouse RA-IL15-Fc GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGSGGGGSGGGGSGGGSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSGGGSEPKS SDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK SEQ ID NO: 25: RA-IL15-Fc ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEPKSSDKT HTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLPGK SEQ ID NO: 26: Mouse IL15-RA-Fc NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINT SSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKS SDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 27: Human IL15-RA-Fc NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINT SSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSS DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 28: mouseRB-L2-IL15-RA-Fc AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVCTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLE MLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTPADPMKEGGGGSPVGLIGGGGSNWIDVRYDLEKIESLIQSIHIDTTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFIN TSSGGGSGGGSGGGSGGGSGGGSGGGSGLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK sequence number 29: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGARYRWLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 30: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGRIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 31: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGAIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 32: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGRAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 33: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGAAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 34: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGRSENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 35: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGASENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 36: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGRPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 37: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGAPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 38: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGLISHSITAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 39: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGNLRSKLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 40: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGVFSIPLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 41: RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTWSPWSQPLAFRTKPALGKDTGGGGSSGIKYHSLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGSGGGSGGGSGGGSGGGSGLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:42: RB-L1-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0238] The disclosure of the application is described herein in preferred embodiments with reference to figures depicting the same or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Thus, appearances of "in one embodiment," "in an embodiment," and similar language herein may, but do not necessarily, all refer to the same embodiment.

[0239] The features, structures, or characteristics described in the disclosure of the application may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present invention. However, those skilled in the relevant art will recognize that the applicant's compositions and / or methods may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the disclosure.

[0240] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Although similar or equivalent methods and materials as those described herein can also be used in the practice or testing of this disclosure, preferred methods and materials are now described.The methods described herein can be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0241] <Cited by reference> This disclosure refers to and cites other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, etc. All such documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is said to be incorporated herein by reference but that contradicts existing definitions, statements, or other disclosure materials explicitly set forth herein, is incorporated only to the extent that no contradiction arises between the incorporated material and the material of this disclosure, as explicitly set forth herein. In the event of a conflict, the conflict should be resolved in favor of this disclosure as the priority disclosure.

[0242] <equivalent> The representative examples are intended to help illustrate the invention and are not intended to, and should not be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document, including the examples and references to the scientific and patent literature contained herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

1. A fusion protein comprising: First structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof; Second structural unit: active interleukin 15 (IL15), Third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment L1 covalently bonding the first, second, and third structural units; wherein the second structural unit is located between the first structural unit and the third structural unit; The fusion protein comprises: a fourth structural unit: the extracellular domain of the IL15 receptor β subunit (RB), located at the N-terminus of the fusion protein; further comprising a linker segment L2 covalently linking the fourth structural unit and the remaining structural units of the fusion protein; wherein the first structural unit is covalently bonded to the C-terminus of the fourth structural unit; and a fusion protein, wherein the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

2. A fusion protein comprising: First structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof; Second structural unit: active interleukin 15 (IL15), Third structural unit: antibody Fc fragment, and a first linker segment L1 covalently bonding the first, second, and third structural units; wherein the second structural unit is located between the first structural unit and the third structural unit; The fusion protein comprises: A fourth structural unit: the extracellular domain of the IL15 receptor β subunit (RB), further comprising a linker segment L2 covalently linking the fourth structural unit and the remaining structural units of the fusion protein; wherein the first structural unit is covalently bonded to the fourth structural unit; and a fusion protein, wherein the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

3. The fusion protein of claim 2, wherein the fourth structural unit is located at the C-terminus of the fusion protein and the first structural unit is covalently bound to the N-terminus of the fourth structural unit.

4. A fusion protein comprising: First structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof; Second structural unit: active IL15, Third structural unit: an antibody Fc fragment located at the C-terminus of the fusion protein; and a first linker segment L1 covalently bonding the first, second, and third structural units; wherein the second structural unit is located at the N-terminus of the fusion protein, and the first structural unit is located between the second structural unit and the third structural unit; The fusion protein comprises: a fourth structural unit: the extracellular domain of the IL15 receptor β subunit (RB), located at the N-terminus of the fusion protein; further comprising a linker segment L2 covalently linking the fourth structural unit and the remaining structural units of the fusion protein; wherein the second structural unit is covalently bonded to the C-terminus of the fourth structural unit; and a fusion protein, wherein the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

5. A fusion protein comprising: First structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof; Second structural unit: active IL15, Third structural unit: antibody Fc fragment, and a linker segment L1 covalently bonding the first, second, and third structural units; wherein the first structural unit is located between the second structural unit and the third structural unit; The fusion protein comprises: A fourth structural unit: the extracellular domain of the IL15 receptor β subunit (RB), further comprising a linker segment L2 covalently linking the fourth structural unit and the remaining structural units of the fusion protein; wherein the second structural unit is covalently bonded to the fourth structural unit; and a fusion protein, wherein the linker segment L2 is recognizable and hydrolyzable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

6. The fusion protein of claim 5, wherein the fourth structural unit is located at the C-terminus of the fusion protein and the second structural unit is covalently bound to the N-terminus of the fourth structural unit.

7. The fusion protein of any one of claims 1 to 6, wherein the IL15R subunit is selected from an α subunit, a β subunit, and a γ subunit.

8. The fusion protein of claim 7 , wherein the IL15R subunit is an α subunit.

9. The fusion protein of any one of claims 1 to 7, wherein the fragment is the sushi domain of the α subunit of IL15R having the amino acid sequence set forth in SEQ ID NO:

4.

10. The fusion protein according to any one of claims 1 to 7, wherein the IL15 is human or mouse IL15.

11. The fusion protein of claim 10, wherein the IL15 is mouse IL15.

12. The fusion protein of claim 11, wherein the mouse IL15 has the amino acid sequence set forth in SEQ ID NO:

1.

13. The fusion protein of any one of claims 1 to 12, wherein the antibody Fc fragment comprises a human Fc fragment.

14. The fusion protein of claim 13, wherein the antibody Fc fragment comprises a human IgG1-Fc having the amino acid sequence set forth in SEQ ID NO:

3.

15. The fusion protein of any one of claims 1 to 14, wherein the linker segment L1 comprises a plurality of GGGS.

16. The fusion protein of any one of claims 1 to 14, wherein the first linker segment attached to the third structural unit comprises the amino acid sequence set forth in SEQ ID NO:

9.

17. The fusion protein according to any one of claims 1 to 14, wherein the linker segment L1 connecting the first and second structural units comprises the amino acid sequence set forth in SEQ ID NO:

8.

18. The fusion protein according to any one of claims 1 to 6, wherein the amino acid sequence of the RB has the amino acid sequence set forth in SEQ ID NO:

6.

19. The fusion protein of any one of claims 1-6 or 18, wherein the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

20. 20. The fusion protein of claim 19, wherein the matrix metalloproteinase is matrix metalloproteinase 9 (MMP9).

21. 20. The fusion protein of claim 19, wherein the matrix metalloproteinase is matrix metalloproteinase 14 (MMP14).

22. The fusion protein of any one of claims 1-6 or 18-21, wherein the linker segment L2 comprises an amino acid sequence set forth in SEQ ID NO: 10-23.

23. A homodimeric or heterodimeric protein comprising a fusion protein according to any one of claims 1 to 21.

24. 24. The homodimeric or heterodimeric protein of claim 23, RA-IL15-Fc monomer: A homodimeric or heterodimeric protein comprising a fusion protein of the sushi domain of the IL15 receptor α subunit, a linker segment L1, mouse IL15, a linker segment L2, and human IgG1 Fc, wherein the amino acid sequence of the RA-IL15-Fc monomer has the amino acid sequence set forth in SEQ ID NO:

24.

25. 24. The homodimeric or heterodimeric protein of claim 23, IL15-RA-Fc monomer: A homodimeric or heterodimeric protein comprising a fusion protein of mouse IL15, a linker segment L1, the sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1 Fc, wherein the amino acid sequence of the IL15-RA-Fc monomer has the amino acid sequence set forth in SEQ ID NO:

26.

26. 24. The homodimeric or heterodimeric protein of claim 23, IL15-RA-Fc monomer: A homodimeric or heterodimeric protein comprising a fusion protein of human IL15, a linker segment L1, the sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1 Fc, wherein the amino acid sequence of the IL15-RA-Fc monomer has the amino acid sequence set forth in SEQ ID NO:

27.

27. 24. The homodimeric or heterodimeric protein of claim 23, RB-IL15-RA-Fc monomer: A homodimeric or heterodimeric protein comprising a fusion protein of the extracellular domain of the IL15 receptor β subunit, a linker segment L2, mouse IL15, a linker segment L1, the sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1 Fc, wherein the amino acid sequence of the RB-IL15-RA-Fc monomer has the amino acid sequence set forth in SEQ ID NO:

28.

28. 24. The homodimeric or heterodimeric protein of claim 23, RB-IL15-RA-Fc monomer: A homodimeric or heterodimeric protein comprising a fusion protein of the extracellular domain of the IL15 receptor β subunit, a linker segment L2, human IL15, a linker segment L1, the sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1 Fc, wherein the amino acid sequence of the RB-IL15-RA-Fc monomer has the amino acid sequence set forth in SEQ ID NOs: 29-41.

29. A homodimeric or heterodimeric protein according to any one of claims 24 to 28, which is hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

30. 30. A substantially purified protein according to any one of claims 1-29.

31. A polynucleotide encoding the protein of any one of claims 1 to 30.

32. 32. An expression vector comprising the polynucleotide of claim 31.

33. A pharmaceutical composition comprising a protein according to any one of claims 1 to 30 and a pharmaceutically acceptable excipient, carrier, or diluent.

34. 31. Use of a protein according to any one of claims 1-30 and a pharmaceutically acceptable excipient, carrier or diluent in the preparation of a medicament for treating or ameliorating a disease or disorder.

35. 35. The use of claim 34, wherein the disease or disorder is selected from head and neck cancer, endometrial cancer, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain cancer, stomach cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

36. Use of a protein according to any one of claims 1 to 30 for the preparation of a medicament.

37. 37. The use of claim 36, wherein the agent is an antitumor agent.

38. 38. The use of claim 37, wherein the anti-tumor agent is effective for treating B-cell lymphoma or for anti-colon cancer.

39. A cell line comprising a polynucleotide encoding the protein of any one of claims 1-30.

40. 40. A process for producing a protein comprising culturing the cell line of claim 39.

41. 41. The process of claim 40, further comprising purifying or isolating the produced protein.

42. 1. A method for producing a protein, comprising: Providing an expression vector encoding the protein of any one of claims 1 to 30; introducing the expression vector into a host cell; culturing said host cells in a medium under conditions sufficient to express said protein, and purifying said protein from said host cells or the medium. A method for producing a protein.

43. 43. The method of claim 42, wherein the host cell is selected from a 293F cell and a CHO cell.

44. 44. The method of claim 42 or 43, wherein the introduction of the expression vector is by transient transfection.

45. 45. The method of any one of claims 42-44, wherein the protein is purified by Protein A / G affinity chromatography or size exclusion.

46. 46. ​​An isolated protein produced by the method of any one of claims 42-45.

47. 47. The isolated protein of claim 46, which is substantially pure.

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