Interleukin 15 fusion protein, and its composition and treatment method

Abstract

The present invention provides novel fusion proteins of interleukin-15 and prodrugs, as well as their compositions and methods of preparation, useful in the treatment of various diseases and disorders (eg, hyperplasia, solid tumors, or hematopoietic malignancies). [Selection diagram] None

Description

Technical Field

[0001] Priority Claim and Related Applications This application claims the benefit of Chinese Application No. 201810420739.6, filed on May 4, 2018, the entire content of which is incorporated herein by reference for all purposes.

[0002] Technical Field of the Invention The present invention generally relates to novel fusion proteins and their therapeutic use. More specifically, the present invention provides novel fusion proteins of interleukin 15 and prodrugs, as well as their compositions and methods of preparation that are useful for the treatment of various diseases and disorders (e.g., hyperplasia, solid tumors or hematopoietic malignancies).

Background Art

[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). Similar to interleukin 2, IL15 belongs to the family of 4 - helix bundle cytokines. The human IL15 gene has been mapped to the region of chromosome 4 q25 - 35. Mature IL15 consists of 112 amino acids and contains three N - glycosylation sites. The expression of IL15 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, but the mature protein is mainly produced by dendritic cells, monocytes, macrophages, stromal cells, and not by T cells. The expression of IL - 15 is stimulated by cytokines such as granulocyte - macrophage colony - stimulating factor (GM - CSF), interferon, and agonists of toll - like receptor (TLR) (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 proteins. Both IL2Rα and IL15Rα contain conserved sushi domains. IL15 has some functions similar to 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 mainly expressed in dendritic cells (DCs) and monocytes. In most cases, IL15 binds to the receptor in a trans - presenting manner. In the trans - presentation 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. Next, IL15Rα can trans - present IL - 15 to responding cells such as T cells and NK cells.

[0006] IL15 exhibits the following multifaceted functions in homeostasis and the activation of both innate and adaptive immunity. (1) IL15 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) IL15 plays an important role in the activation, proliferation, and differentiation of DCs by the autocrine model, promotes the expression of MHC II and CD80 / CD86 on DCs, and increases the presentation of DCs to CD8 + T cells, (6)IL15 plays an important role in the activation of monocytes and macrophages, (7)IL15 plays an important role in the inhibition of AICD, protects T cells from inhibition by Tregs, and overcomes resistance to tumor antigens.

[0007] IL2 has been approved by the FDA for the treatment of metastatic renal cell carcinoma and malignant melanoma. However, the effectiveness of IL-2 as an anti-cancer therapeutic agent has been questioned due to the maintenance of CD4 + CD25 + T regulatory cells and its very important role 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 the therapeutic effect.

[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. Recent reports have shown that the administration of a pre-formed complex of IL15 and its soluble receptor IL15Rα extends 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 succin domain and IL15 linked by a flexible peptide showed an extended half-life of IL15 and improved 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. Moreover, IL15 showed enhanced anti-tumor effects or inhibition of tumor growth 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] There are various side effects associated with IL15 therapy, for example, (1) Induction of cytokine cascades including TNFα, IL1, IL6, GM-CSF and pro-inflammatory cytokines (2) Promotion of 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 molecules PD1 / PDL1 That is.

[0011] For example, currently available treatments and methods for hyperplasia, solid tumors or hematopoietic malignancies are inadequate. There remains an urgent and continuing need for new and improved treatments for effectively treating 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. It is disclosed herein that novel fusion proteins of IL15 and its prodrugs, compositions thereof and methods of preparation are useful in treating various diseases and disorders, such as hyperplasia, solid tumors or hematopoietic malignancies.

[0013] In one aspect, the present invention relates generally to a fusion protein. The fusion protein comprises 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) that covalently binds to the first, second and third structural units, with the first structural unit 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. The fusion protein comprises a first structural unit: a subunit or fragment thereof of interleukin 15 receptor alpha (IL15Rα), 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) that covalently binds to 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 structural unit and the third structural unit.

[0015] In yet another aspect, the present invention relates generally to a fusion protein. The fusion protein comprises a first structural unit: a subunit or fragment thereof of interleukin 15 receptor alpha (IL15Rα), 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 or fragment thereof of interleukin 15 receptor beta (IL15Rβ), and a first linker segment (L1) that covalently binds to 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 generally to a fusion protein. The fusion protein comprises a first structural unit: a subunit or fragment thereof of interleukin 15 receptor (IL15R), 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 or fragment thereof of interleukin 15 receptor beta (IL15Rβ), a first linker segment (L1) that covalently binds to the first, second, and third structural units, and a second linker segment (L2) that the fourth structural unit covalently binds to the second structural unit, wherein the first structural unit is located between the second structural unit and the third structural unit and the N-terminus of the fusion protein is the fourth structural unit.

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

[0018] In yet another aspect, the present invention relates generally to a fusion protein disclosed herein or a substantially purified protein such as a fragment.

[0019] In yet another aspect, the present invention relates generally to a polynucleotide encoding a protein such as a fusion protein disclosed herein or a fragment thereof.

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

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

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

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

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

[0025] In yet another aspect, the present invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein or a fragment thereof disclosed herein, for treating or alleviating a disease or disorder (e.g., hyperplasia, solid tumor or hematopoietic malignancy).

[0026] In yet another aspect, the present invention relates generally to the use of a protein, such as a fusion protein or a 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., hyperplasia, solid tumor or hematopoietic malignancy).

[0027] In yet another aspect, the present invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein or a 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., hyperplasia, solid tumor or hematopoietic malignancy).

[0028] In yet another aspect, the present invention relates generally to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein or a fragment thereof disclosed herein.

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

[0030] In yet another aspect, the present invention generally relates to a method of producing a protein. The method includes providing an expression vector encoding a protein such as a fusion protein or a 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.

[0031] In yet another aspect, the present invention generally relates to an isolated protein produced by the process disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0032]

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[0033] Definitions 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 in the context in which they are found.

[0034] When trade names are used herein, the trade names include product formulations, generic pharmaceuticals, and pharmaceutical active ingredients of the named products, unless otherwise indicated by the context.

[0035] The ranges provided herein are to be understood as encompassing all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-ranges 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 1" is understood to be 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] 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 apparent from the context, the term "about" is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean. About is understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise apparent from the context, all numerical values provided herein can be modified by the term "about".

[0040] As used herein, unless specifically stated or apparent from the 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 "consisting essentially of" shall mean that the compositions and methods include the recited elements and exclude any other elements that are optional and essentially important for the compositions and methods. For example, "consisting essentially of" refers to administering a pharmacologically active agent explicitly recited and excludes pharmacologically active agents not explicitly recited. The term consisting essentially of does not exclude pharmacologically inactive or inert agents, such as pharmaceutically acceptable excipients, carriers or diluents. The term "consisting of" shall mean, when used to define compositions and methods, excluding trace amounts of other components and substantial method steps. Embodiments defined by each of these transitional 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 produce a cellular response. An agonist can be a ligand that binds directly to the receptor. Alternatively, an agonist can bind indirectly to the receptor, for example, by (a) forming a complex with another molecule that binds directly to the receptor or (b) otherwise effecting a modification of another compound such that the other compound binds directly 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 and thereby blocks the action of the agonist or inverse agonist at the receptor. However, the 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. This term is meant to include whole antibodies and antigen-binding fragments thereof. This term encompasses polyclonal, monoclonal, chimeric, Fab, Fv, single-chain antibodies, and one or more immunoglobulin variable chains, or CDR domain designs, as well as bispecific and multispecific antibodies. The antibody can be derived from any animal. Preferably, the antibody can be derived from a mammal, such as a human, mouse, rabbit, goat, guinea pig, camel, horse, etc., or other suitable animal. The antibody can recognize a polypeptide or polynucleotide antigen. This term includes, for example, antigen-binding fragments of immunoglobulins, variable and / or constant regions of heavy chains, variable and / or constant regions of light chains, complementarity-determining regions (cdr), and active fragments containing framework regions. This term includes preparations of polyclonal antibodies and monoclonal antibodies, as well as 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; preparations containing minibodies, humanized antibody molecules, and any functional fragments retaining specific binding obtained from such molecules.

[0045] As used herein, the term "antigen" as used herein means any substance that causes the immune system to generate an antibody or a specific cellular immune response thereto. A disease-related antigen is any substance related to any disease that causes the immune system to generate an antibody or a specific cellular immune response thereto. An antigen can be recognized by the immune system and / or can induce a humoral immune response and / or a 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 typically in a highly selective manner with its corresponding antibody or TCR and does not react with many other antibodies or TCRs that can be induced by other antigens. The antigen as used herein can also be a mixture of several individual antigens.

[0046] As used herein, the term "biologically active" entity, or an entity having "biological activity" means any entity having 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 can generate a desired effect. Biological activity can include an improved desired activity or a decrease in an undesired activity. For example, an entity exhibits biological activity when it participates in a molecular interaction with another molecule, has therapeutic value in alleviating a medical condition, has prophylactic value in inducing 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 can be synthesized from known elements, such as recombinant synthesis or chemical synthesis, and can contain heterologous elements.

[0047] As used herein, the terms "cancer" and "cancerous" refer to, or describe, a physiological state in a mammal characterized typically by unregulated cell growth. Examples of cancers include, but are not limited to, carcinomas, lymphomas, sarcomas, blastomas, and leukemias. 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, eukaryote, primary cell or immortalized cell line, or any group of such cells, such as a tissue or an organ. Preferably, the cell is of mammalian (e.g., human) origin and can be infected by one or more pathogens.

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

[0050] As used herein, the term "co-expressed" refers to the simultaneous expression in a host cell of two different polypeptides such that the two polypeptides can interact or bind and form a complex in either the host cell or 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 being distinct from a state of health or normalcy. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[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 compounds of the present invention can 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 the nucleic acid molecule into a gene product. The gene product can be the direct transcript of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or other types of RNA), or a peptide or polypeptide produced by translation of the mRNA. The gene product also includes RNA 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 any recipient of a recombinant vector or isolated polynucleotide, or an individual cell or cell culture that was a recipient. 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 it can be a cell of any origin that can be used to increase the nucleic acids described herein. Host cells include the progeny of a single host cell, and the progeny are not necessarily identical to the original parent cell (in morphology or total DNA complement) due to natural, accidental, or intentional mutations and / or changes. Host cells include cells transfected or infected in vivo or in vitro with the recombinant vectors or polynucleotides of the present invention. A host cell containing a recombinant vector of the present invention can 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, cells of gram-positive bacteria such as those of the genus Bacillus, Streptomyces, and Staphylococcus, and cells of gram-negative bacteria such as those of the genus 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, cells of Drosophila and Sf9 cells. Plant cells include, inter alia, cells from crops such as cereals, medicinal or ornamental plants or bulbs. Mammalian cells suitable for the present invention include epithelial cell lines (such as pigs), osteosarcoma cell lines (such as humans), neuroblastoma cell lines (such as humans), epithelial carcinomas (such as humans), glial cells (such as mice), hepatocyte cell lines (such as monkeys), CHO cells (Chinese hamster ovary), COS cells, BHK cells, HeLa cells, 911, AT1080, A549, 293 or PER., C6, human ECC NTERA-2 cells, D3 cells of the mESC line, HS293 and BGV01, human embryonic stem cells such as 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 that includes the sequence of the non-antigen binding fragment of the whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any of the immunoglobulins (e.g., IgG1, IgG2). Native Fc consists of monomeric polypeptides that can be linked by covalent (i.e., disulfide bonds) and non-covalent bonds into a dimeric or multimeric form. The number of intermolecular disulfide bonds between the monomeric subunits of the native Fc molecule ranges from 1-4 depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgAl, IgGA2).

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

[0058] "Fc domain" includes native Fc as defined herein, as well as Fc variant molecules and sequences. Similar to Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from the whole antibody or produced by recombinant gene expression or other means.

[0059] Fc fusion proteins have been reported to combine the Fc region of IgG with domains of 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. Patent Nos. 7,754,855; 5,480,981; 5,808,029; WO 7 / 23614; WO 98 / 28427, and the references cited therein). Fc fusion proteins can include mutant Fc molecules (such as those described in U.S. Patent No. 7,732,570). Fc fusion proteins can be soluble in plasma or can bind to the cell surface of cells having specific Fc receptors.

[0061] As used herein, the term "Fc variant" refers to a molecule or sequence that has been altered from native Fc but still contains a binding site for the salvage receptor, FcRn. International applications WO97 / 34631 (published September 25, 1997) and WO96 / 32478 describe exemplary Fc variants and their interaction with the salvage receptor, and are incorporated herein by reference. Thus, the term "Fc variant" includes molecules or sequences that have been humanized from non-human native Fc. Furthermore, native Fc contains sites that can be removed as they provide structural features or biological activities that are not required for the fusion molecules of the present invention. Thus, in certain embodiments, the term "Fc variant" refers to a molecule or sequence that lacks 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 the salvage receptor, or (7) antibody-dependent cell cytotoxicity (ADCC). Fc variants are described in more detail below.

[0062] As used herein, the term "fusion protein" refers to a polypeptide that contains two or more regions from different or heterologous proteins that have been covalently linked (i.e., "fused") by recombinant, chemical, or other suitable means. 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 assist 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) deoxyribonucleosides, 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%) more than the highest recommended dose of a particular compound for the treatment of human diseases or conditions.

[0065] As used herein, the term "immune response" refers to a process by which immune cells are stimulated and / or replenished from the blood into lymphoid and non-lymphoid tissues via a multi-factorial process involving distinct adhesion and / or activation steps. Activation conditions cause the release of cytokines, growth factors, chemokines, and other factors, up-regulate 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 generation of memory cell types. Immune response also means the activity of immune cells that suppress or regulate 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 terms "identical" or percent "identity" in the context of two or more nucleic acid or polypeptide sequences refer to sequences that are identical or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., the IL15 or IL15Rα sequence) 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 algorithms with the 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 be applied with respect to, the complement of a test sequence. The definition includes sequences having deletions and / or additions, as well as sequences having substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over a region of at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over a region of 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the full length of an amino acid or nucleic acid sequence.

[0067] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, sub-sequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence based on the program parameters.

[0068] Preferred examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST algorithms 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 (ncbi.nlm.nih.gov / ) of the National Center for Biotechnology Information. Either default parameters or other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults an 11 wordlength (W), an expectation (E) of 10, M = 5, N = -4, and comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a 3 wordlength and an expectation (E) of 10, and a 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 comparison of both strands.

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

[0070] As used herein, the terms "interleukin 15" or "IL15" refer to a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the native mammalian IL15 amino acid sequence that is biologically active, i.e., the mutant protein ("mutant") has a function (more than 75%) similar 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 the balance between this IL15 and IL2. IL15 induces the activation of JAK kinases and the phosphorylation and activation of the transcription activators STAT3, STAT5, and STAT6. IL15 also increases the expression of the apoptosis inhibitor BCL2L1 / BCL-X(L), probably via the transcriptional activation activity of STAT6, and thus blocks apoptosis. Two alternative splicing transcriptional variants of the IL15 gene encoding the same mature protein have been reported.

[0072] Exemplary functional assays of the IL15 polypeptide include the proliferation of T cells (e.g., Montes el al. 2005 Clin Exp immunol 142:292), and the activation of NK cells, macrophages, and neutrophils. Methods for isolating specific immune cell subsets and detecting proliferation (i.e., 3 the incorporation of H thymidine) are well known in the art. Cell-mediated cytotoxicity assays can be used to measure the activation of NK cells, macrophages, and neutrophils. Isotopes ( 51Cell-mediated cytotoxicity assays, including the release of chromium (Cr), dyes (e.g., tetrazolium, neutral red), or enzymes, are also well known in the art, along 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). For example, apoptosis assays, including the TUNEL assay and the annexin V assay, are well known in the art, along with commercially available kits (R&D Systems, Minneapolis, Minn) (e.g., Coliga et el. 1991-2006 Current Methods in Immunology John Wiley & Sons.).

[0073] As used herein, the term "interleukin-15 receptor alpha" or "IL15Rα" refers to the interleukin-15 receptor alpha amino acid sequence derived from a mammal. Those skilled in the art recognize that the interleukin-15 receptor alpha nucleic acid and amino acid sequences are publicly available on GenBank via gene databases, such as the World Wide Web of the National Center for Biotechnological Information (ncbi.nlm.nih.gov). Exemplary native mammalian IL-15 receptor alpha nucleic acid or amino acid sequences can be derived from, for example, humans, primates, dogs, cats, pigs, horses, cows, sheep, rodents, mice, rats, hamsters, guinea pigs, etc. Accession numbers of exemplary native mammalian IL-15 nucleic acid sequences include NM_172200.1 (human isoform 2), and NM_002189.2 (human isoform 1 precursor). Accession numbers of 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 that is biologically active and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native mammalian IL15Rα amino acid sequence, having a function (more than 75%) similar to that of the native IL15Rα protein in at least one functional assay. IL15Rα is a cytokine receptor that specifically binds to IL-15 with high affinity. One functional assay is specific binding to native IL-15 protein.

[0075] As used herein, the term "isolated" molecule (such as a polypeptide or polynucleotide) refers to a molecule that has been engineered to exist at a higher concentration than in nature or has been removed from its native environment. For example, an antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, or 20%, or 40%, or 50%, or 70%, 90% of non-antibody material that it is not naturally bound to has been removed. For example, a polynucleotide or polypeptide that naturally exists in a living animal is not "isolated", but the same polynucleotide or polypeptide separated from its native coexisting substances is "isolated". Further, a recombinant DNA molecule contained in a vector is considered isolated for the purposes of the present invention. Isolated RNA molecules include DNA molecules and in vivo or in vitro RNA replication products of RNA molecules. Isolated nucleic acid molecules further include synthetically generated molecules. In addition, vector molecules contained in recombinant host cells are also isolated. Thus, 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 may enable the connected molecules or groups to acquire a functional arrangement. A 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] Elements of a fusion protein, such as cytokines or other bioactive molecules, and any peptide linkers, can be organized in almost any manner provided that the fusion protein has the intended function. In particular, each element of the fusion protein can be spaced from another element by at least one appropriate peptide linker segment or sequence, if desired. Additionally, the fusion protein can include tags, for example, to facilitate modification, identification, and / or purification of the fusion protein. More specific fusion proteins are described in the following examples.

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

[0079] As used herein, the term "medium" or "media" includes any culture medium, solution, solid, semi-solid, or rigid support that can support or contain a bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, CHO cell, prokaryotic host cell, E. coli, or Pseudomonas host cell, and any host cell containing cellular contents. Thus, the term can include the medium in which the host cell has grown, for example, the medium in which a polypeptide has been secreted, including the medium either before or after the growth step. The term can also include buffers or reagents containing host cell lysates, such as when the polypeptide is produced intracellularly and the host cell is 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 blockade 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 with, or blocks, the measured activity of a polypeptide or polynucleotide when compared to an appropriate control. For example, a "modulator" can bind and / or activate or inhibit a target with measurable affinity or can 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. Nucleic acid sequences that are operably linked need not be physically adjacent 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 the 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 medium such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material involved in carrying or transporting the subject pharmaceutical 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 injurious 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 its derivatives such as carboxymethylcellulose sodium, ethyl cellulose, and cellulose acetate; tragacanth powder; 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; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; physiological saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymers, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and fragrances, 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 of any length, including ribonucleotides and deoxyribonucleotides. They can include both double-stranded, single-stranded, or triple-stranded sequences, including cDNA, mRNA, viral (e.g., DNA viruses and retroviruses), or genomic DNA sequences from prokaryotic and eukaryotic cell sources; RNAi; cRNA; antisense molecules; recombinant polynucleotides; ribozymes; and synthetic DNA sequences, but are not limited to these. 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 polynucleotides as they occur in nature, but also include polynucleotides in which unnatural nucleotide analogs and inter-nucleotide linkages occur. Nucleic acid molecules can include modified nucleic acid molecules (e.g., modified bases, sugars, and / or nucleotide linkers). Non-limiting examples of this type of unnatural structure include polynucleotides in which the sugar is different from ribose, polynucleotides in which phosphodiester linkages 3'-5' and 2'-5' occur, polynucleotides in which reverse linkages (3'-3' and 5'-5') and branched structures occur. Also, the polynucleotides of the present invention include non-natural inter-nucleotide linkages such as peptide nucleic acids (PNA), locked nucleic acids (LNA), methylphosphonates, phosphoramidates, C1-C6 alkyl phosphotriesters, phosphorothioate and phosphorodithioate type C1-C4 alkyl phosphonate linkages. In any case, the polynucleotides of the present invention maintain the ability to hybridize to target nucleic acids in a manner similar to natural polynucleotides.

[0085] Unless otherwise indicated or not apparent from the context, a particular nucleic acid sequence also implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), and complementary sequences, as well as the explicitly recited sequences. 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 bases 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 methods for precluding, delaying, avoiding, or halting the onset, occurrence, severity, or recurrence of a disease or condition. For example, a method is considered a prevention if there is a decrease or delay in the onset, occurrence, severity, or recurrence of a disease or disorder or one or more symptoms thereof in a subject susceptible to the disease or condition as compared to a subject not undergoing the method. The disclosed methods are also considered a prevention if there is a decrease 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 undergoing the method as compared to the progression of the subject prior to receiving treatment. A decrease or delay in the onset, occurrence, severity, or recurrence of osteoporosis can be about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of decrease therebetween.

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

[0088] As used herein, the term "promoter" refers to a DNA regulatory region that can bind RNA polymerase in mammalian cells and initiate transcription of a downstream (3' direction) coding sequence operably linked thereto. A promoter sequence contains the minimum number of bases or elements necessary to initiate transcription of a gene of interest at a level higher than background. A promoter sequence may contain a transcription start site, as well as protein-binding domains (consensus sequences) involved in the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain a "TATA" box and a "CAT" box. Promoters include those that are naturally adjacent to a nucleic acid molecule and those that are not naturally adjacent to a nucleic acid molecule. Further, the term "promoter" includes inducible promoters, conditional active promoters such as cre-lox promoters, constitutive promoters, and tissue-specific promoters.

[0089] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, etc. 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, etc. Further, a polypeptide can refer to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, as long as the protein maintains the desired activity. These changes can be intentional or accidental. Amino acids can be referred to herein by either the commonly known three-letter symbols or 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 of components normally associated with or interacting with the protein in its natural environment, i.e., natural cells, or in the case of recombinantly produced proteins, as found in the host cell. A protein that may be substantially free of cellular material includes preparations of contaminating protein that are 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). 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 the dry weight of the 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 a purity level of 99% or more, as determined by appropriate methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0091] The proteins and prodrugs of the present invention, following their preparation, are preferably isolated and / or purified to obtain a composition containing them in an amount of 80% by weight or more ("substantially pure"), which is then used or formulated as described herein. In certain embodiments, the compounds of the present invention are of a purity greater than 95%.

[0092] As used herein, the term "receptor" refers to a protein, including a glycoprotein or a fragment thereof, that is capable of interacting 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, when bound to a receptor, usually initiates a cellular response such as the initiation of a signal transduction pathway. A receptor does not necessarily have to be 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, semi-synthetic, and / or synthetic origin, which is not bound to some or all of the polynucleotide to which it is naturally bound due to its origin or manipulation. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by the expression of a recombinant polynucleotide. The term "recombinant" as used 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, an animal, or a 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 removing it from a human or an animal, or it can be tested in vitro. A sample can be tested after treatment, for example, by histological methods. A "sample" also refers to a cell, including a liquid or tissue sample, or a cell separated from a liquid or tissue sample. A "sample" is also a newly collected cell, tissue, organ, or liquid from a human or an animal, or a processed or preserved cell, tissue, organ, or liquid.

[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, such as 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 an aqueous solution at a temperature above about 5-37°C, at or near neutral pH, in the presence or absence of a low concentration of an anionic or nonionic surfactant. Under these conditions, the sedimentation value of a soluble protein is often low, e.g., less than about 10-50 Svedberg units.

[0096] The aqueous solutions referred to herein typically have a buffer compound to establish the pH, typically within a pH range of about 5-9, and an ionic strength range of about 2 mM to 500 mM. A protease inhibitor or a mild nonionic surfactant may be added. Additionally, a carrier protein (e.g., bovine serum albumin) can be added if desired. Exemplary aqueous buffers include standard phosphate buffered saline, Tris buffered saline, or other well-known buffers and cell culture formulations.

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

[0098] As used herein, the term "stimulate" or "stimulation" refers to increasing, amplifying, enhancing, boosting a physiological activity, such as an immune response. The stimulation can result in 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). The subject can be a mammal. The term "mammal" or "mammalian" refers to any animal within the taxonomic classification of mammals. Mammals can be human or non-human mammals, such as dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice. 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 "suppress" or "suppressing" refers to reducing, weakening, diminishing, halting, or stabilizing a physiological activity, such as an immune response. Suppression can be a negative change. For example, the reduction can be 5%, 10%, 25%, 50%, 75%, or even up to 90 - 100%. Exemplary reductions 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 the dosage of a therapeutic agent (s)ufficient 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 initially administering a low dose of the agent and then gradually increasing the dosage 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 facilitator 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 terms "treatment" or "treating" of a disease or disorder refer to ways of reducing, delaying, or improving such a condition, or one or more symptoms of such a disease or condition, either before or after it occurs. Treatment can be directed at one or more effects or symptoms of the disease and / or the underlying pathological condition. Treatment can be any reduction and can be complete ablation of the disease or symptoms of the disease, but is not limited thereto. 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 cells.

[0105] As used herein, the term "vector" refers to a nucleic acid molecule capable of transmitting genetic material to a host cell or organism. Vectors are composed of either DNA or RNA. Vectors have an origin of replication of their own, one or more unique recognition sites for restriction endonucleases that can be used for the insertion of foreign DNA, and conventional selectable markers such as genes encoding antibiotic resistance, and often recognition sequences (such as promoters) for the 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 a novel fusion protein and its therapeutic use. More specifically, the present invention provides a novel fusion protein of IL15 and its prodrug, its composition and preparation method, which are useful for treating various diseases and disorders, such as hyperplasia, solid tumors or hematological malignancies, with reduced off-target toxicity and side effects during treatment.

[0108] In one aspect, the present invention generally relates to a fusion protein. The fusion protein comprises a first structural unit: a subunit or fragment thereof of the interleukin 15 receptor (IL15R); 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 at the N-terminus of the fusion protein and the second structural unit is located between the first structural unit and the third structural unit.

[0109] In another aspect, the present invention generally relates to a fusion protein. The fusion protein comprises a first structural unit: a subunit or fragment thereof of the interleukin 15 receptor (IL15R); 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, and a first 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 the α subunit, β subunit, and γ subunit.

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

[0112] In certain embodiments of the fusion protein, the fragment is the sushi domain of the α subunit of murine IL15R having the amino acid sequence set forth in SEQ ID NO: 4.

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

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

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

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

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

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

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

[0120] In certain embodiments of the fusion protein, the linker segment L1 that links the first structural unit to the third structural unit comprises the amino acid sequence set forth in SEQ ID NO: 9.

[0121] In certain embodiments of the fusion protein, the linker segment L1 that connects the first and second structural units comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0122] In certain embodiments, 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 that covalently links the fourth structural unit to the remaining structural units of the fusion protein, wherein the first structural unit is covalently attached 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 hydrolysable by proteolytic enzymes specifically expressed in the tumor microenvironment.

[0123] In certain embodiments, 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 that covalently links the fourth structural unit to the remaining structural units of the fusion protein, the second structural unit is covalently attached 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 hydrolysable by proteolytic enzymes specifically expressed in the tumor microenvironment.

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

[0125] In certain embodiments, 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 shown in SEQ ID NOs: 10-23.

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

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

[0132] In certain embodiments, the homodimeric protein or heterodimeric protein comprises a fusion protein of a monomer of RA-IL15-Fc: the sushi domain of the human IL15 receptor α subunit, linker segment L1, human IL15, linker segment L1, and a human IgG1 Fc having, for example, the amino acid sequence set forth in SEQ ID NO: 25.

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

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

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

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

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

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

[0139] In yet another aspect, the present invention relates generally to a substantially purified protein, such as a fusion protein or fragment, disclosed herein.

[0140] In yet another aspect, the present invention relates generally to a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein.

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

[0142] In yet another aspect, the present invention relates generally to a pharmaceutical composition 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 present invention relates generally 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 relates generally to a method for treating a disease or condition. The method comprises 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 hyperplasia, solid tumor, or 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 subjecting the subject to radiation therapy.

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

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

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

[0154] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), sunitinib (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 (Lonafamib) (SCH 66336), sorafenib (Sorafenib) (BAY43-9006, Bayer Labs), and gefitinib (Gefitmib) (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and CYTOXAN® cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, uredopa; ethylene imines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylmelamine; acetogenins (especially bratasin and bratasinone); camptothecin (including synthetic analog topotecan); bryostatin; calicheamicin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogs); cryptophycins (especially cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin;Nitrogen mustards such as chlorambucil, chromafazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobenbitin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, particularly calicheamicin gamma1 and calicheamicin omega1 (Angew Chem.Intl.Ed.Engl.(1994)33:183-186); dynemicin including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), actinomycin, autramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esonarubicin, idarubicin, marcellomycin, mitomycin such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, keramycin, rhodrubicin, streptozocin, streptozocin, tubercidin, ubenimex, dinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimethoprim; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, etc.;Androgens such as calusterone, drostanolone propionate, epithioestanol, mepitiostane, and testolactone; anti-adrenal agents such as aminoglutethimide, mitotane, and trilostane; folic acid supplements such as folic acid (frolic acid); aceglatone, aldophosphamide glycoside, aminolevulinic acid, eniluracil; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demeclocycline; diacozine; elfomithine; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraerine; pentostatin, phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazine; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg); razoxane; lysocine; sizofiran; spirogermanium; tenuazonic acid; triazicone, 2,2’,2’’-trichloroethylamine, trichothecene (especially, T-2 toxin, verracurin A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gasitocin; arabinoside (“Ara-C”); chlorambucil; GEMZAR (registered trademark) (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (registered trademark) (vinorelbine);Novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing are included.

[0155] Examples of a second (or additional) agent or therapeutic agent can include, but are not limited to, 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, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, chlorambucil, carmustine, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), intercalating anticancer agents (e.g., actinomycin, anthracycline, bleomycin, mitomycin C, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide, etc.), immunotherapy agents (e.g., interleukin, interferon, 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, for treating or alleviating a disease or disorder (e.g., hyperplasia, solid tumor or hematopoietic malignancy).

[0157] In yet another aspect, the present invention relates generally to the use of a polynucleotide encoding a protein, such as a fusion protein or a fragment thereof disclosed herein, for treating or alleviating a disease or disorder (e.g., hyperplasia, solid tumor or hematopoietic malignancy).

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

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

[0160] In certain embodiments, the medicament 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, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

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

[0163] In yet another aspect, the present invention relates generally to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein or a fragment thereof disclosed herein.

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

[0165] In yet another aspect, the present invention relates generally to a method for producing a protein, the method comprising providing an expression vector encoding a protein, such as a fusion protein or a 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, such as 293F and CHO cells.

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

[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 present invention relates generally to an isolated protein produced by the method disclosed herein.

[0171] In certain embodiments, the 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 from about 7 to 20 amino acids, such as from 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 undesirable conformation. The linker sequence can be used, for example, to space recognition sites from the fused molecule. Specifically, the peptide linker sequence can be arranged to provide flexibility to the molecule. The linker preferably consists mainly of amino acids having small side chains such as glycine, alanine, and serine to provide flexibility.

[0174] Overall, the preparation of the fusion protein complex of the present invention can be accomplished by the procedures disclosed herein and by recognized recombinant DNA techniques including, for example, polymerase chain reaction (PCR), plasmid DNA preparation, DNA cleavage with restriction enzymes, oligonucleotide preparation, DNA ligation, mRNA isolation, introduction of DNA into appropriate cells, transformation or transfection of hosts, and culturing of hosts. Further, the fusion molecule can be isolated and purified using chaotropic agents and well-known methods of electrophoresis, centrifugation, and chromatography. (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 sequences and DNA sequences encoding the fusion proteins to be presented. 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 substantial amounts of the fusion proteins or their components. The DNA sequences can be inserted into appropriate expression vectors, i.e., vectors containing the elements necessary for transcription and translation of the inserted protein coding sequences. A variety of host-vector systems are available for expressing the protein coding sequences. These include mammalian cell lines infected with viruses (e.g., vaccinia virus, adenovirus, etc.); insect cell lines 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 related 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 cassette format. The term "cassette" means that each component can be readily replaced with another by standard recombinant methods. In particular, DNA vectors constructed in cassette format are particularly desirable when the encoded fusion complex is to be used against pathogens that may express or have a serotype.

[0177] To prepare a vector encoding a fusion protein complex, the sequence encoding a biologically active polypeptide is ligated to the sequence encoding an effector peptide by using an appropriate ligase. The DNA encoding a presentation peptide can be obtained from natural resources such as appropriate cell lines or by isolating DNA by known synthetic methods, such as the phosphotriester method (OligonuclEOTIDE SYNTHESIS, IRL PRESS, M.J. GAIT, ED., 1984). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. Once isolated, the gene encoding a biologically active polypeptide can be amplified by PCR or other means known in the art. Appropriate PCR primers for amplifying the gene encoding a biologically active polypeptide can add restriction sites to the PCR product. The PCR product preferably contains splice sites for the effector peptide and leader sequences necessary for appropriate expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably contains a sequence encoding a linker sequence or restriction enzyme sites for ligation of such sequences.

[0178] The fusion proteins described herein can be generated 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 ligated to the DNA sequence encoding the effector polypeptide or, more typically, a DNA sequence encoding a linker sequence discussed herein is inserted between the sequence encoding the biologically active polypeptide and the sequence encoding the effector polypeptide and can be ligated using an appropriate ligase. The resulting hybrid DNA molecule can be expressed in a suitable host cell to generate a fusion protein complex. The DNA molecules are ligated to each other in a 5'-3' orientation such that the translation frame of the encoded polypeptides is not altered after the ligation reaction (i.e., the DNA molecules are ligated to each other in-frame). The resulting DNA molecule encodes an in-frame fusion protein.

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

[0180] When obtaining a biologically active polypeptide of a variant, an IL15, an IL15R or an Fc domain coding sequence, one of ordinary skill in the art will recognize that the polypeptide can be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, conservative amino acid substitutions, i.e., substituting one amino acid for another of similar size, charge, polarity, and conformation, are well known to be unlikely to significantly change the function of a protein. The 20 standard amino acids that make up proteins can be roughly classified into four groups of conservative amino acids as follows: 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. Substituting one amino acid in a protein for another within the same group is unlikely to adversely affect the biological activity of the protein. In other examples, modifications can be made at amino acid positions to reduce or enhance the biological activity of a protein. Such changes can be introduced randomly or via site-directed mutagenesis based on the known or putative structural or functional properties of the target residue. Following the expression of the mutant protein, changes in biological activity due to the modification can be readily evaluated using binding or functional assays.

[0181] The homology between nucleotide sequences can be determined by DNA hybridization analysis, where the stability of the double-stranded DNA hybrid depends on the degree of base pair formation present. Conditions of high temperature and / or low salt content can decrease the stability of the hybrid and can be varied to prevent annealing of sequences with less than a selected degree of homology. For example, for a sequence with a G-C content of about 55%, hybridization and washing conditions of 40-50°C, 6×SSC (sodium chloride / sodium citrate buffer), and 0.1% SDS (sodium dodecyl sulfate) indicate about 60-70% homology, hybridization and washing conditions of 50-65°C, 1×SSC, and 0.1% SDS indicate about 82-97% homology, and hybridization and washing conditions of 52°C, 0.1×SSC, and 0.1% SDS indicate about 99-100% homology. A wide variety of computer programs are also available for comparing (and measuring the degree of homology of) nucleotide sequences with amino acid sequences. 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 an appropriate vector by known methods by using restriction enzymes to create a cut in the vector for subsequent insertion of the construct followed by a ligation reaction. Next, the vector containing the gene construct is introduced into a host suitable for expression of the fusion protein. (Sambrook et. al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989) for disclosure relating to these methods.)

[0183] The selection of an appropriate vector can be done empirically based on factors related to the cloning protocol. For example, the vector needs to have appropriate response elements for the host being used. Further, the vector must be able to accommodate the DNA sequence encoding the expressed fusion protein complex. Suitable host cells include eukaryotic and prokaryotic cells, preferably cells that can be easily transformed in a culture medium and can exhibit rapid growth. Specifically, preferred host cells include prokaryotes such as Escherichia coli (E. coli), Bacillus subtilis, and eukaryotes such as animal cells and yeast strains, for example, Saccharomyces cerevisiae (S. cerevisiae). Mammalian cells are generally preferred, especially J558, NSO, SP2-O or CHO. Other suitable hosts include, for example, insect cells such as Sf9. Conventional culture conditions are employed. See Sambrook, supra. Stable transformed or transfected cell lines can be selected. Cells expressing the fusion protein complex of the present invention can be determined by known procedures. For example, the expression of a fusion protein complex bound to an immunoglobulin can be determined by ELISA and / or immunoblotting specific for the bound immunoglobulin. Other methods for detecting the expression of a fusion protein containing a biologically active polypeptide bound to the IL12 or IL12R domain are disclosed in the examples.

[0184] Host cells can be used for the preparation purpose of propagating nucleic acids encoding a desired fusion protein or its components. Host cells can include prokaryotic or eukaryotic cells in which the production of the fusion protein is particularly intended. Thus, host cells include, in particular, yeast, flies, worms, plants, frogs, mammalian cells and organs capable of propagating nucleic acids encoding fusions. 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 capable of propagating nucleic acids encoding a desired fusion protein complex also include non-mammalian eukaryotic cells, including insects (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha, which are generally reviewed by Fleer, R., 1992 Current Opinion in Biotechnology, 3(5):486496). Also intended are certain prokaryotes such as E. coli and Bacillus.

[0186] Nucleic acids encoding a desired fusion protein 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, virus introduction and / or integration.

[0187] Various promoters (transcription start regulatory regions) can be used according to 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 selection of a 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. Overexpression of a protein can be harmful to the host cell. As a result, the growth of the host cell can be restricted. By using an inducible promoter system, the host cell can be cultured to an acceptable density prior to induction of gene expression, facilitating a higher product yield.

[0189] Various signal sequences may be used according to the present invention. Signal sequences homologous to the biologically active polypeptide coding sequence may be used. Alternatively, signal sequences selected or designed for efficient secretion and processing within the expression host may also be used. The signal sequence can be directly linked to the protein coding sequence through a sequence encoding a signal peptidase cleavage site or 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 employed to join two fragments of DNA. Polylinkers and adapters can be used to facilitate the joining of the selected fragments. Expression constructs can typically be assembled at the stage where rounds of E. coli restriction, ligation, and transformation are employed. A number of cloning vectors suitable for the construction of expression products are known in the art (λlZAR and pBLUESCRIPT SK-l, Stratagene, La Jolla, Calif., pET, Novagen Inc., Madison, Wis., rEE12.4, Lonza Biologies Basel, Switzerland).

[0191] The expression construct may be transformed into a host as a cloning vector construct, or it can be used in a linear or circular form, removed from a 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 the desired fusion protein. In that process, a nucleic acid encoding the protein of interest operably linked to a control sequence is introduced into a host cell (e.g., yeast, fungus, insect, bacterial, or animal cell), and it is grown at production scale in a culture medium to stimulate the transcription of the nucleotide sequence encoding the desired fusion protein. Subsequently, the desired fusion protein is isolated from the 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 the harvested cells. In particular, the purification techniques can be used to express and purify the desired fusion protein on a large scale (i.e., at least in milligram amounts) from various setups 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 then the supernatant is purified by affinity or immunoaffinity chromatography. For example, protein A or protein G affinity chromatography or immunoaffinity protocols involve the use of monoclonal antibodies that bind to the expressed fusion complex, such as the linked TCR or immunoglobulin region. The fusion proteins of the present invention can be separated 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, 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 reverse phase high performance liquid chromatography, methods that utilize differences in isoelectric point such as isoelectric focusing electrophoresis (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 protein of the present invention is preferably substantially pure. That is, it is separated from the accompanying cellular substituents such that the fusion protein is present with at least 80% or 90%-95% homogeneity (w / w). A fusion protein having at least 98-99% homogeneity (w / w) is 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 purified to partial or substantial purity, the soluble fusion protein is disclosed herein for therapeutic use or in performing in vitro or in vivo assays. Substantial purity can be determined by various 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, a fusion protein, a polynucleotide, a gene construct, a vector or host cell according to the invention and a pharmaceutically acceptable excipient or solvent.

[0196] Preferred excipients for use in the present invention include sugars, starches, celluloses, gums and proteins. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated in a pharmaceutical form for administration as a solid (e.g., tablets, capsules, troches, granules, suppositories, crystalline or amorphous sterile solids that can be reconstituted to provide a liquid form such as solutions, suspensions, emulsions, elixirs, lotions, ointments, etc.) or semi-solid (gels, ointments, creams and the like). The pharmaceutical composition of the present invention can be administered by any route, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intracranial, ventricular, percutaneous, subcutaneous, intraperitoneal, intranasal, enteral, topical, subdermal or rectal routes. Modifications of different forms of effective principle administration, the excipients used and their manufacturing procedures can be found in Remington's Pharmacy (A.R. Gennaro, Ed.), 20 thedition, Williams & Wilkins PA, USA (2000) Examples of pharmaceutically acceptable solvents are known in the state of the art and include phosphate, water, emulsions such as emulsions, different types of wetting agents, and aqueous saline solutions buffered with sterile solutions, etc. The compositions containing said solvents can be formulated by conventional procedures known in the state of the art.

[0197] In the case of the pharmaceutical composition of the present invention containing a nucleic acid (the polynucleotide, vector or gene construct of the present invention), the present invention contemplates a specially prepared pharmaceutical composition for administering said nucleic acid. The pharmaceutical composition has the advantage of administering the nucleic acid naked, i.e., eliminating the toxicity associated with the reagents used for transfection of compounds that protect the nucleic acid from degradation by nucleases of organisms. Suitable routes of administration for the naked compound include intravascular, intratumoral, intracranial, intraperitoneal, splenic, intramuscular, subcutaneous, mucosal, topical and oral routes (Templeton, 2002 DNA Cell Biol., 21:857-867) or administering the nucleic acid to the forming part of liposomes, conjugating it to cholesterol, or conjugating it to a compound that can promote translocation through cell membranes such as the TAT peptide derived from the TAT protein of HIV-1, or the third helix of the homeodomain of the antennapedia protein of D. melanogaster, the VP22 protein of herpes simplex virus, oligomers of arginine, and peptides such as those described in WO07069090 (Lindgren, et al. 2000 Trends PharmacolSci 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 to a retrovirus such as a part forming a plasmid vector or a viral vector, preferably an adenovirus-based vector, an adeno-associated virus, or a retrovirus such as a murine leukemia virus (MLV) or a lentivirus (HIV, FIV, EIAV).

[0198] The composition 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 the agent, in other words, about 4.4×10 16 copies 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. The single dose can be administered by injection, inhalation or topical administration. The bifunctional polynucleotides and compositions of the present 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 can vary from a few days to several months or until the condition recurs. The optimal dosage can be determined by periodically measuring the concentration of the agent in the patient's organism. The optimal dosage can be determined from the EC50 value obtained through previous in vitro or in vivo tests in animal models. This unit dosage can be administered once a day or less than once a day, preferably less than once every 2, 4, 8 or 30 days. Alternatively, an initial dosage can be administered followed by one or more maintenance dosages, which are generally in an amount less than the initial dosage. The maintenance regimen may include treating the patient with dosages in the range of 0.01 μg - 1.4 mg / kg 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 dosage is preferably administered at most once every 5, 10 or 30 days. Treatment must be continued for a period of time that varies depending on the type of change the patient undergoes, its severity and the patient's condition. After treatment, the evolution of the patient must be monitored to determine whether the dosage should be increased in the case of a disease that does not respond to treatment or decreased if improvement of the disease or undesirable secondary effects are observed.

[0200] The daily dosage can be administered as a single dose or in more than one dose, depending on the particular situation. If repeated or frequent administration is required, it is recommended to implant an administration device such as a pump, a semi-permanent catheter (intravenous, intraperitoneal, intima or intracapsular) or a reservoir.

[0201] The compositions of the present invention are administered according to methods known to experts in the art and include, but are not limited to, intravenous, oral, nasal, parenteral, topical, transdermal, rectal and the like.

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

Example

[0203] The following examples illustrate specific 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 of skill in the art, can be applied to the compounds, subclasses, and species as disclosed herein.

[0204] Example 1. Construction of Fusion Proteins A. Construction of Four Fusion Proteins Construction of Control Protein: IL15-Fc Mouse IL15 is 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 as Figures 1B and 1C. Similarly in biological activity, both structures are interchangeably referred to as Super IL15. The amino acid sequence of mouse IL15Rα sushi was SEQ ID NO:4. The amino acid sequence of human IL15Rα sushi was SEQ ID NO:5. The sequence of the linker between IL15Rα sushi and IL15 was 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 linked by linker segment L2.

[0207] A schematic diagram of IL15RβECD-L2-IL15-IL15Rα sushi-Fc (named 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. The 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 MMP14 substrate linker amino acid sequence is SEQ ID NO: 11-23.

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

[0209] The detailed protocol is as follows.

[0210] Construction of the fusion protein IL15, IL15RA and IL15RB ECD were synthesized and cloned into the pEE12.4-IgGκ-hIgG1 Fc plasmid, which contained mouse IgGκ as the leader sequence and human IgG1 Fc. The plasmid was extracted using a standard commercial plasmid extraction kit and stored at -80°C.

[0211] Transfection of the fusion protein 293F cells were cultured in CD OptiCHO™ medium and incubated at 37°C, 8% CO 2 in an incubator with shaking at 135 rpm. Two days before transfection, the cells were plated at a density of 0.6 - 0.8×10 6 cells / mL. The cells were about 2.5 - 3.5×10 6Cells were collected at a density of 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. DNA and PEI were mixed and incubated at room temperature for 5 minutes, and then mixed with the cells in the flask. The flask was placed in an incubator at 37 °C with 8% CO 2 while shaking at 85 rpm. 200 mL of EX-CEFF (TM) 293 medium was added 4 hours after transfection at 135 rpm. 3.8 mM VPA was added 20 hours after transfection. The supernatant was collected from day 4 to day 7 after transfection while the cell viability was over 70%.

[0212] Purification of the fusion protein 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: 0.1 M glycine, pH 2.7 Regeneration buffer: 1 M NaOH Neutralization buffer: 1 M Tris-HCl, pH 9.0 All buffers were filter-filtered through a 0.45 μm filter. (1) The sample was centrifuged at 8000 × rpm for 2 hours to remove cells, and then filtered through a 0.45 μm filter. NaN 3 was added to a final concentration of 0.05% to prevent bacterial growth. (2) When the column was stored with 20% ethanol, the column was washed with distilled water at a linear flow rate of 50 to 100 cm / h for 5 column volumes. (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 protein 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 first characterized by its ability to stimulate the proliferation of the mouse T cell line CTLL - 2. In this protocol, CTLL - 2 cells are cultured in the presence of serial dilutions of mouse IL - 15, and their proliferation is measured by CCK8.

[0215] The following procedure was used. (1) CTLL - 2 assay medium supplemented with 100 U / mL recombinant human IL - 2 was used to culture CTLL2 cells. (2) CTLL - 2 cells were collected during the logarithmic growth phase 24 - 48 hours after sub - culturing, washed twice to remove residual IL - 2. Cells were resuspended in 5 - 10 mL of CTLL - 2 assay medium, the cells were counted, and adjusted to a concentration of 2×10 4 cells / mL. (3) Samples were diluted using CTLL - 2 assay medium. The initial top concentration was 10 μg / mL. Serial 1:10 dilutions were made on 7 tubes. (4) 100 μL of the cell suspension was added to each flat - bottom 96 - well plate (2×10 3 cells / well). 100 μL of each sample was added to each well. A row of wells containing only 200 μL of assay medium was included as a negative control. (5) The plate was covered and incubated for 48 - 72 hours. 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 shows that (1) the biological activities of the two forms of super IL-15 are similar, that is, in the fusion protein, first, regardless of the IL-15 fragment or the IL-15α sushi fragment, the function of super IL-15 was not affected, and (2) the biological activity of super IL-15 increased by approximately 100-fold compared with IL-15-Fc.

[0217] B. The fusion fragment of IL-15RβECD can block the biological function of super IL-15 The proliferative abilities of mouse RB-IL15-RA-Fc and super IL-15 on CTLL2 were examined by CCK8 assay. The results shown in Figure 4 indicate that the biological activity of RB-IL15-RA-Fc decreased by 100-fold. This shows that the extracellular domain of IL-15Rβ can block the biological function of super IL-15.

[0218] C. Antitumor effects and systemic toxicity in different tumor models A20 model Experiment 1 (25 μg): A20 cells (3×10 6 ) were subcutaneously injected into the right flank of Balb / c mice. Mice with tumors (60 - 80 mm 3 ) were treated intratumorally (i.t.) and intravenously (i.v.) with 25 μg of super IL-15 on days 10 and 13. The control group was treated with PBS. Tumor volume = length × width × height / 2. The tumor growth curve was recorded.

[0219] Results: (1) In the intratumoral treatment group, tumors regressed completely in all mice (Figure 5A). Mice that had already undergone complete tumor regression were rechallenged with a lethal dose of A20 cells. All mice rejected the rechallenged tumors and showed a strong memory response (Figure 5C). (2) All mice died on the second iv treatment, which showed severe systemic toxicity (Figure 5B).

[0220] Experiment 2 (12.5 μg): A20 cells (3×10 6 ) were subcutaneously injected into the right flank of Balb / c mice. Mice with tumors (60 - 80 mm 3 ) were treated intratumorally (i.t.) and intravenously (i.v.) 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 intratumoral treatment group, 100% of the mice had complete regression. In contrast, 20% of the intravenously treated mice had complete regression, and the remaining mouse tumors were partially suppressed.

[0222] MC38 model Experiment: MC38 cells (5×10 5 ) were subcutaneously injected into the right flank of C57 mice. Mice with tumors (60 mm 3 ) were intravenously treated with 25 μg of super-IL15 intratumorally (i.t.) and intravenously (i.v.) 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 intratumoral treatment group, 50% of the mice had complete regression (Figure 6A), and the survival rate increased significantly (Figure 6B). In contrast, intravenously administered mice did not have complete tumor regression (Figure 6A), and the mouse survival rate increased slightly (Figure 6B).

[0224] The above results indicate that super-IL-15 appears to function locally in the tumor microenvironment (TME).

[0225] <Example 3. Comparison of the Tumor Therapeutic Effects and Side Effects of Super-IL-15 and RB-IL15-RA-Fc> A20 cells (3×10 6 ) were subcutaneously injected into the right flank of Balb / c mice. Mice with tumors (60 - 80 mm 3 ) were treated intraperitoneally (i.p.) with 12.5 μg of super-IL-15 or Rβ-IL15-RA-Fc on days 10 and 13. Tumor growth was measured twice a week. Serum was collected 20 hours after the second injection. Cytokine concentrations in the serum were measured by Cytometric Bead Array (CBA), and tumor curves were recorded.

[0226] The following CBA protocol was used. (1) Serum was collected from the ophthalmic vein and stored at -80 °C. (2) IL12p70, IL-6, IFN-γ, TNFα, MCP1, and IL-10 in the serum were evaluated using a CBA kit from BD. (3) Standards were reconstituted with 2.0 mL of assay diluent and then re-calibrated 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) 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)Performed Th1 / Th2 / Th17 cytokine assay: Vortexed the mixed capture beads and added 20 μL to all assay tubes. Added 50 μL of mouse Th1 / Th2 / Th17 cytokine standard diluent to the control tubes. Added 50 μL of each unknown sample to appropriately labeled sample assay tubes. Incubated the assay tubes at room temperature for 2 hours, protected from light. (6)Added 1 mL of wash buffer to each assay tube and centrifuged at 300 g for 5 minutes. (7)Carefully aspirated the supernatant and discarded it from each assay tube. Added 300 μL of wash buffer to each assay tube and resuspended the bead pellet. (8)The samples were analyzed via flow cytometry and the cytokine levels were calculated according to the standard.

[0227] This therapeutic effect is shown in Figure 8A, indicating 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, indicating that the toxic side effects of RB-IL15-RA-Fc are significantly reduced compared to super-IL15.

[0229] A20 cells (3×10 6 ) were subcutaneously injected into the right flanks of Balb / c mice. Mice with tumors (60 - 80 mm 3 ) were treated intraperitoneally (i.p.) with 25 μg of super-IL15 or RB-IL15-RA-Fc on days 10 and 13. Tumor growth was measured twice a week. Serum was collected 20 hours after the second injection. Cytokine levels in the serum were measured by Cytometric Bead Array (CBA) and tumor curves were recorded.

[0230] Results: After tumor rechallenge and treatment with Super IL15, mice with tumors developed significantly into disease with severe weight loss, reduced mobility, and wrinkled fur, and all died within one day after the second treatment. In contrast, none of the mice treated with RB-IL15-RA-Fc died, and none of the mice appeared unhealthy. The survival curve of RB-IL15-RA-Fc was significantly longer than that of Super IL15. The survival curve is shown in Figure 9A, and the 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 were also generated and tested in vitro. The production of human proteins was carried out according to the cloning, transfection, and purification protocols described previously for the production of mouse 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] Figure 10 shows the results of SDS-PAGE electrophoresis of the purified human fusion protein incubated with or without MMP14 at 37°C for 24 hours.

[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 TMIL-2 cells cause activation of STAT5 and secretion of the SEAP subsequence. The level of STAT5-induced SEAP can be easily monitored using QUANTI-Blue™. Since IL15 binds and transmits signals through a complex calibrated from 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) Gently wash the HEK-Blue IL-2 cells in PBS and resuspend them in fresh, pre-warmed test medium (DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated FBS (56 °C, ~1×10 6 cells / mL for 30 min). (2) Serial dilute the samples in a flat-bottom 96-well plate and incubate at 37 °C for 20 - 24 h in a CO 2 incubator with 50 μL of the cell suspension (~50,000 cells) per well. (3) Incubate 20 μL of the induced HEK-BLUE IL-2 cell supernatant per well of the flat-bottom 96-well plate with 100 μL of the resuspended QUANTI-Blue™ solution for 15 min - 1 h in an incubator at 37 °C. (4) Determine the SEAP level using a spectrophotometer at 650 nm.

[0237] Results: Figure 11 demonstrates that RB-L2-15RA-Fc, constructed using the MMP14 substrate sequence embedded in the linker segment (L2) and incubated with MMP14, showed the same level of function as 15RA-Fc regardless of the presence or absence of MMP14. Consistently, RB-L1-15RA-Fc, constructed without the MMP14 substrate sequence embedded in the linker segment (L1), functioned similarly to samples without MMP14. The construct designation 15RA is short for IL15-L1-RA. <Sequence Listing> Accession No. 1: Mouse IL15 NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS Accession No. 2: Human IL15 NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS Accession No. 3: Human IgG1-Fc EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession No. 4: Mouse Rα-Sushi domain GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT Accession No. 5: Human Rα-Sushi domain ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP Accession No. 6: Mouse Rβ extracellular domain AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE SEQ ID NO: 7: Human Rβ extracellular domain AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT SEQ ID NO: 8: Linker segment L1 SGGGSGGGGSGGGGSGGGGSGGGSLQ SEQ ID NO: 9: Linker segment L1 GGGGS SEQ ID NO: 10: Linker segment L2 (MMP9) GGGGS PVGLI GGGGS 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 Accession No. 14: Linker Segment L2 (MMP14) GGGGSSGRAMHMYTAGGGGS Accession No. 15: Linker Segment L2 (MMP14) GGGGSSGAAMHMYTAGGGGS Accession No. 16: Linker Segment L2 (MMP14) GGGGSSGRSENIRTAGGGGS Accession No. 17: Linker Segment L2 (MMP14) GGGGSSGASENIRTAGGGGS Accession No. 18: Linker Segment L2 (MMP14) GGGGSSGRPENIRTAGGGGS Accession No. 19: Linker Segment L2 (MMP14) GGGGSSGAPENIRTAGGGGS Accession No. 20: Linker Segment L2 (MMP14) GGGGSSGLISHSITAGGGGS Accession No. 21: Linker Segment L2 (MMP14) GGGGSSGNLRSKLTAGGGGS Accession No. 22: Linker Segment L2 (MMP14) GGGGSSGVFSIPLTAGGGGS Accession No. 23: Linker Segment L2 (MMP14) GGGGSSGIKYHSLTAGGGGS Accession No. 24: Mouse RA-IL15-Fc GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGGSGGGGSGGGGSGGGSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 25: Human RA-IL15-Fc ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession No. 26: Mouse IL15-RA-Fc NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession No. 27: Human IL15-RA-Fc NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Accession number 28: Mouse RB-L2-IL15-RA-Fc AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKEGGGGSPVGLIGGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK Accession number 29: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGARYRWLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 30: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 31: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 32: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 33: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 34: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRSENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 35: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGASENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 36: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 37: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 38: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGLISHSITAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 39: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGNLRSKLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 40: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGVFSIPLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 41: Human RB-L2-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGIKYHSLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Sequence number 42: Human RB-L1-IL15-RA-Fc AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

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

[0239] The features, structures, or characteristics described in the disclosure of the application can be combined in any suitable manner in one or more embodiments. In the description of this specification, many specific details are set forth in order 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 can 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 in detail or described in order to avoid obscuring aspects of the present disclosure.

[0240] 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. Methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, but the preferred methods and materials are now described. The methods described herein can be performed in any logically possible order in addition to the specific order disclosed.

[0241] <Citation by reference> In the present disclosure, references and citations are made to other documents such as patents, patent applications, patent publications, journals, books, papers, web content, etc. All such documents are hereby incorporated by reference in their entirety for all purposes. Although said to be incorporated by reference herein, materials or portions thereof that are inconsistent with existing definitions, statements, or other disclosure materials explicitly described herein are incorporated only to the extent that no conflict arises between the incorporated materials and the materials of the present disclosure. In the event of a conflict, the present disclosure shall prevail as the prior disclosure to resolve the conflict.

[0242] <Equivalents> The representative examples are intended to assist in explaining the present invention and are not intended to limit, nor are they to be construed as limiting, the scope of the present invention. Indeed, various modifications of the present invention, in addition to those shown and described herein, will become apparent to those of ordinary skill in the art from the entire contents of this document, including the examples and references of the scientific and patent literature included herein. The examples include important additional information, illustrations, and guidance that can be adapted to the practice of the present invention in its various embodiments and their equivalents.

Claims

Claim 1 A fusion protein comprising: A first structural unit that is a subunit of the interleukin-15 receptor (IL15R) or a fragment thereof, wherein the subunit of the IL15R is the α subunit, and the fragment is the sushi domain of the α subunit of the IL15R; A second structural unit that is active interleukin-15 (IL15); A third structural unit that is an antibody Fc fragment located at the C-terminus of the fusion protein; A linker segment L1 that covalently binds to the first, second, and third structural units; A fourth structural unit that is the extracellular domain of the IL15 receptor β subunit (RB) and is located at the N-terminus of the fusion protein; and A linker segment L2 that covalently binds to the fourth and first structural units of the fusion protein, wherein the first structural unit is covalently bound to the C-terminus of the fourth structural unit, the second structural unit is located between the first and third structural units, and the linker segment L2 is recognizable and hydrolysable by proteolytic enzymes specifically expressed in the intratumoral microenvironment. Claim 2 A fusion protein comprising: A first structural unit that is a subunit of the interleukin-15 receptor (IL15R) or a fragment thereof, wherein the subunit of the IL15R is the α subunit, and the fragment is the sushi domain of the α subunit of the IL15R; A second structural unit that is active IL15; A third structural unit that is an antibody Fc fragment located at the C-terminus of the fusion protein; A linker segment L1 that covalently binds to the first, second, and third structural units; A fourth structural unit that is the extracellular domain of the IL15 receptor β subunit (RB) and is located at the N-terminus of the fusion protein; and A linker segment L2 that covalently binds to the fourth and second structural units of the fusion protein, wherein the second structural unit is covalently bound to the C-terminus of the fourth structural unit, the first structural unit is located between the second and third structural units, and the linker segment L2 is recognizable and hydrolysable by proteolytic enzymes specifically expressed in the intratumoral microenvironment. Claim 3 The fusion protein according to claim 1 or 2, wherein the IL15 is human IL15.

4. The fusion protein according to claim 1 or 2, wherein the IL15 is mouse IL15.

5. The fusion protein according to claim 4, wherein the mouse IL15 has the amino acid sequence set forth in SEQ ID NO:

1.

6. The fusion protein according to claim 5, wherein the antibody Fc fragment comprises a human Fc fragment.

7. The fusion protein according to claim 6, wherein the antibody Fc fragment comprises human IgG1-Fc having the amino acid sequence set forth in SEQ ID NO:

3.

8. The fusion protein according to any one of claims 1-7, wherein the linker segment L1 comprises a plurality of GGGGS.

9. The fusion protein according to any one of claims 1-7, wherein the linker segment L1 linked to the third structural unit comprises the amino acid sequence set forth in SEQ ID NO:

9.

10. The fusion protein according to any one of claims 1-7, wherein the linker segment L1 linking the first and second structural units comprises the amino acid sequence set forth in SEQ ID NO:

8.

11. The fusion protein according to claim 1 or 2, wherein the amino acid sequence of the RB has the amino acid sequence set forth in SEQ ID NO:

6.

12. The fusion protein according to claim 11, wherein the protease specifically expressed in the intratumoral microenvironment is a matrix metalloprotease.

13. The fusion protein according to claim 12, wherein the matrix metalloprotease is matrix metalloprotease 9 (MMP9).

14. The fusion protein according to any one of claims 11-13, wherein the linker segment L2 comprises the amino acid sequences set forth in SEQ ID NOs: 10-23.

15. A homodimeric protein or a heterodimeric protein comprising the fusion protein according to any one of claims 1-14.

16. A monomer of RB-IL15-RA-Fc, which is a fusion protein of the extracellular domain of the IL15 receptor β subunit, linker segment L2, mouse IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, and human IgG1 Fc, and has the amino acid sequence set forth in SEQ ID NO: 28, or A monomer of RB-IL15-RA-Fc, which is a fusion protein of the extracellular domain of the IL15 receptor β subunit, the linker segment L2, human IL15, the linker segment L1, the sushi domain of the IL15 receptor α subunit, the linker segment L1, and human IgG1 Fc, and has the amino acid sequences set forth in SEQ ID NOs: 29-41 The fusion protein according to claim 2, comprising at least one of the above.

17. The homodimeric protein or heterodimeric protein according to claim 15, which is hydrolyzed by a proteolytic enzyme specifically expressed in the intratumoral microenvironment.

18. The protein according to any one of claims 1-17, which is substantially purified.

19. A polynucleotide encoding the protein according to any one of claims 1-18.

20. An expression vector comprising the polynucleotide according to claim 19.

21. A pharmaceutical composition comprising the protein according to any one of claims 1-18, and a pharmaceutically acceptable excipient, carrier, or diluent.

22. The pharmaceutical composition according to claim 21, for use in the treatment of a disease or condition selected from hyperplasia, solid tumor, or hematopoietic malignancy.

23. Use of the protein according to any one of claims 1-18 for the preparation of a medicament for the treatment of a disease or condition selected from hyperplasia, solid tumor, or hematopoietic malignancy.

24. The use according to claim 23, wherein the disease or disorder is selected from head and neck cancer, endometrial cancer, colorectal cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

25. A cell line comprising a polynucleotide encoding the protein according to any one of claims 1-18.

26. A method for producing a protein, comprising the step of culturing the cell line according to claim 25.

27. The method according to claim 26, further comprising the step of purifying or isolating the produced protein.

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