Interleukin-15 fusion protein, composition and treatment method thereof

Abstract

The present invention provides novel interleukin-15 fusion proteins and prodrugs thereof, compositions and preparation methods that can be used to treat various diseases and disorders (eg, hyperplasia, solid tumors or hematopoietic malignancies).

Description

[0001] Priority claim and related applications

[0002] This application claims the benefit of Chinese Application No. 201810420739.6, filed on May 4, 2018, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The present invention generally relates to novel fusion proteins and their therapeutic uses. More specifically, the present invention provides novel fusion proteins of interleukin 15 and their prodrugs, compositions and methods of preparation that can be used to treat a variety of different diseases and disorders (e.g., hyperplasia, solid tumors or hematopoietic malignancies). Background Art

[0004] Interleukin 15 (IL15), a 14–15 kDa glycoprotein, is a soluble cytokine first discovered in 1994 (Grabstein et al., 1994 Science 264:965-8). Similar to interleukin 2, IL15 belongs to the four-helix bundle cytokine family. The human IL15 gene is mapped to region q25–35 of chromosome 4. Mature IL15 consists of 112 amino acids and contains three N-glycosylation sites. The expression of IL15 is tightly regulated. Although IL-15 mRNA can be found in many tissues and cells including fibroblasts, myocytes, keratinocytes, renal cells, lymphocytes, mast cells, and tumor cells, the mature protein is produced primarily by dendritic cells, monocytes, macrophages, and stromal cells but not by T cells. The expression of IL-15 is stimulated by cytokines such as granulocyte-macrophage colony stimulating factor (GM-CSF), interferons, and agonists of Toll-like receptors (TLRs) (Marek et al., 2011 Cytokine & Growth Factor Reviews 22:99-108).

[0005] The IL15 receptor (IL15R) belongs to the hematopoietic superfamily. The heterotrimeric IL15R contains α, β (CD122) and γ (CD132, common γ chain, γc) subunits. The β subunit (IL15Rβ) is shared with the IL2 receptor. Human IL15Rα belongs to the type I transmembrane protein. Both IL2Rα and IL15Rα contain a conserved sushi domain. IL15 has some functions similar to IL2, such as promoting the proliferation of T and NK cells [3] (Thomas et al., 2006J of Immunology 177:6072-6080).

[0006] IL15Rα is mainly expressed in dendritic cells (DC) and monocytes. In most cases, IL15 binds to the receptor in a trans-presentation form. In the trans-presentation model, IL15 and IL15Rα are synthesized in the same cell. IL15 and IL15Rαsushi domains bind to each other with high affinity in the cytoplasm and transport IL-15 to the cell membrane. Then IL15Rα can trans-present IL-15 to responding cells such as T cells and NK cells.

[0007] IL15 exhibits the following pleiotropic functions in both homeostasis and activation of innate and adaptive immunity:

[0008] (1) IL15 plays an important role in the activation, proliferation and survival of CD8+ T cells;

[0009] (2) IL15 plays an important role in the activation and homeostasis of memory CD8+ T cells;

[0010] (3) IL15 plays an important role in the development, activation, and proliferation of NK cells and NKT cells;

[0011] (4) IL15 plays an important role in the production of anti-tumor antibodies;

[0012] (5) IL15 plays an important role in the activation, proliferation, and differentiation of DCs through an autocrine model, promoting the expression of MHCII and CD80 / CD86 on DCs and enhancing the presentation of DCs to CD8 + T cells;

[0013] (6) IL15 plays an important role in the activation of monocytes and macrophages; and

[0014] (7) IL15 plays an important role in the inhibition of AICD, protecting T cells from Treg inhibition and overcoming resistance to tumor antigens.

[0015] IL2 has been approved by the FDA for the treatment of metastatic renal cell carcinoma and malignant melanoma. + CD25 + The effectiveness of IL-2 as an anticancer therapeutic has been questioned due to its critical role in the maintenance and activation-induced cell death (AICD) of T-regulatory cells. This process leads to the elimination of stimulated T cells and the induction of T-cell tolerance, thus limiting the therapeutic effect.

[0016] Unlike IL2, IL15 does not participate in activation-induced cell death (AICD) and the maintenance of regulatory T cells. Therefore, IL15 may have significant advantages in treating cancer compared to IL2. Recent reports show that the administration of a preformed complex of IL15 and its soluble receptor IL15Rα increases the half-life of IL15 and improves the proliferation of T and NK cells (Thomas et al., 2006 J of Immunology 177: 6072-6080).

[0017] Importantly, the soluble fusion protein of IL15Rαsushi domain and IL15 connected by a flexible peptide showed improved half-life of IL15 and proliferation of T and NK cells. In the mouse B16F10 and DEN-induced HCC tumor models, this fusion protein can inhibit tumor growth and inhibit tumor metastasis. In addition, IL15 showed enhanced anti-tumor effects or inhibited 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).

[0018] A variety of side effects have been associated with IL15 therapy, such as:

[0019] (1) Inducing cytokine cascades including TNFα, IL1, IL6, GM-CSF, and proinflammatory cytokines;

[0020] (2) Promote the proliferation, survival and metastasis of certain tumor cells;

[0021] (3) Activate autoimmune T cells and participate in autoimmune diseases;

[0022] (4) inducing coronary heart disease; and

[0023] (5) Induce the expression of inhibitory molecules PD1 / PDL1.

[0024] Currently available therapeutic agents and methods for, for example, hyperplasia, solid tumors or hematopoietic malignancies are inadequate. There remains an urgent and ongoing need for new and improved therapeutic agents that are effective in treating such diseases and disorders. Summary of the invention

[0025] The present invention is based in part on the surprising discovery of novel fusion proteins and their therapeutic uses. Disclosed herein are novel fusion proteins of IL15 and prodrugs thereof, compositions and methods of making thereof that are useful in treating a variety of different diseases and disorders such as hyperplasia, solid tumors or hematopoietic malignancies.

[0026] In one aspect, the present invention generally relates to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of interleukin 15 receptor α (IL15Rα) or a fragment thereof; a second structural unit: active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; and a first connector segment (L1), which covalently connects 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.

[0027] On the other hand, the present invention generally relates to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of interleukin 15 receptor α (IL15Rα) or a fragment thereof; a second structural unit: active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; and a first connector segment (L1), which covalently connects the first, second and third structural units, wherein the second structural unit is at the N-terminus of the fusion protein, and the first structural unit is located between the second structural unit and the third structural unit.

[0028] On the other hand, the present invention generally relates to a fusion protein. The fusion protein comprises a first structural unit: a subunit of interleukin 15 receptor α (IL15Rα) or a fragment thereof; a second structural unit: active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; a fourth structural unit: a subunit of interleukin 15 receptor β (IL15Rβ) or a fragment thereof; and a first connector segment (L1) which covalently connects the first, second, third and fourth structural units, wherein the fourth structural unit is at the N-terminus of the fusion protein, the second structural unit is located between the fourth and first structural units, and the first structural unit is located between the second structural unit and the third structural unit.

[0029] On the other hand, the present invention generally relates to a fusion protein. The fusion protein comprises: a first structural unit: a subunit or a fragment thereof of interleukin 15 receptor (IL15R); a second structural unit: active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; a fourth structural unit: a subunit or a fragment thereof of interleukin 15 receptor β (IL15Rβ); a first linker segment (L1) which covalently connects the first, second and third structural units, wherein the first structural unit is located between the second structural unit and the third structural unit, and a second linker segment (L2) covalently connects the fourth structural unit to the second structural unit, wherein the fourth structural unit is at the N-terminus of the fusion protein.

[0030] In another aspect, the present invention generally relates to a homodimeric or heterodimeric protein comprising a fusion protein disclosed herein.

[0031] In another aspect, the present invention generally relates to a substantially purified protein, such as a fusion protein or fragment disclosed herein.

[0032] In another aspect, the present invention generally relates to a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

[0033] In another aspect, the present invention generally relates to an expression vector comprising a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

[0034] In another aspect, the present invention generally relates 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.

[0035] In another aspect, the present invention generally relates 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.

[0036] In another aspect, the present invention generally relates to a method for treating a disease or condition comprising 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 tumors, or hematopoietic malignancies.

[0037] In another aspect, the present invention generally relates to the use of a protein, such as a fusion protein disclosed herein, or a fragment thereof, for treating or ameliorating a disease or disorder (eg, a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0038] In another aspect, the present invention generally relates to the use of polynucleotides encoding proteins, such as the fusion proteins disclosed herein, or fragments thereof, for treating or ameliorating a disease or disorder (eg, hyperplasia, solid tumors, or hematopoietic malignancies).

[0039] In another aspect, the present invention generally relates to the use of a protein, such as a fusion protein or fragment thereof disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent for the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., hyperplasia, solid tumors or hematopoietic malignancies).

[0040] In another aspect, the present invention generally relates to the use of polynucleotides encoding proteins, such as the fusion proteins disclosed herein, or fragments thereof, and pharmaceutically acceptable excipients, carriers or diluents for the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., hyperplasia, solid tumors or hematopoietic malignancies).

[0041] In another aspect, the present invention generally relates to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof.

[0042] In another aspect, the present invention generally relates to a method for producing a protein, the method comprising culturing the cell line. In certain embodiments, the method further comprises purifying or isolating the produced protein, such as the fusion protein disclosed herein or a fragment thereof.

[0043] In another aspect, the present invention generally relates to a method for producing a protein. The method comprises: providing an expression vector encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof; introducing the expression vector into a host cell; culturing the host cell in a culture medium under conditions sufficient to express the protein; and purifying the protein from the host cell or the culture medium.

[0044] In another aspect, the present invention generally relates to an isolated protein produced by the methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Schematic diagram showing the structure of the fusion protein. Figure 1 A shows a schematic diagram of the structure of IL15-Fc. Figure 1 B and Figure 1 C shows a schematic diagram of two IL15 superagonists (both referred to as Super IL15): RA-IL15-Fc and IL15-RA-Fc. Figure 1 D shows a schematic diagram of the fusion protein RB-IL15-RA-Fc.

[0046] Figure 2 Exemplary SDS-PAGE electropherograms of three fusion proteins are shown.

[0047] Figure 3 Exemplary results of lymphocyte proliferation assays performed by IL15-Fc and Super IL15 are shown.

[0048] Figure 4 Exemplary results of lymphocyte proliferation assays performed by RB-IL15-RA-Fc and Super IL15 are shown.

[0049] FIG. 5 shows exemplary results of the therapeutic effect of super IL15 in the A20 tumor model. Figure 5A Exemplary data showing the therapeutic effect of super IL15 by intratumoral injection; Figure 5B Exemplary data showing the survival of mice following intratumoral and intraperitoneal injections; Figure 5CExemplary data are shown for tumor-cured mice re-challenged with A20 tumor cells.

[0050] FIG. 6 shows exemplary results of the therapeutic effect of super IL15 in the MC38 tumor model. Fig. 6A Exemplary data of the therapeutic effect of super IL15 by intratumoral and intravenous injection are shown. Figure 6B Exemplary data showing the survival rate of mice after treatment are shown.

[0051] Figure 7 Exemplary data showing the therapeutic effect of super IL15 in the A20 mouse model when a lower dose was administered are shown.

[0052] FIG. 8 shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and Super IL15 in the A20 tumor model following intravenous injection. Fig. 8A Exemplary tumor growth curves of mice after treatment are shown. Figure 8B Exemplary levels of cytokines in serum after treatment are shown.

[0053] FIG. 9 shows an exemplary comparison of the therapeutic effects of RB-IL15-RA-Fc and Super IL15 in the mouse A20 tumor model following intraperitoneal injection. Fig. 9A Exemplary survival rates of mice following treatment are shown. Fig. 9B Exemplary levels of cytokines in serum after treatment are shown.

[0054] Fig.10 Exemplary SDS-PAGE electrophoresis of purified human IL15 fusion proteins digested with or without MMP14 are shown. RB-IL15-RA-Fc is shown as RB-L1-15RA-Fc or RB-L2-15RA-Fc to emphasize whether it is L1 or L2 that is used as the linker segment attached to RB. IL15-RA-Fc is shown as 15RA-Fc.

[0055] Fig.11 Shown is the use of HEK-Blue TM Exemplary results of IL2 reporter cell assay assessing the activity of human IL15 fusion protein incubated with or without MMP14.

[0056] definition

[0057] 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 the invention belongs. The following terms, unless otherwise indicated according to the context in which the terms appear, are intended to have the following meanings.

[0058] When a trade name is used herein, unless the context indicates otherwise, the trade name includes product dosage forms, generic drugs, and active pharmaceutical ingredients of the trade name product.

[0059] The ranges provided herein are understood to be shorthand for all values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from 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.

[0060] As used herein, "at least" a particular value is understood to mean that value and all values ​​greater than that value.

[0061] As used herein, "greater than 1" is understood to mean 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.

[0062] In this specification and the following claims, singular forms include plural referents unless the context clearly dictates otherwise.

[0063] Unless otherwise specified or apparent from the context, when used herein, the term "about" is understood to be within the normal tolerance range in the art, such as within 2 standard deviations of the mean value. Approximately can be 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. All numerical values ​​provided herein can be modified with the term about, unless apparent from the context otherwise.

[0064] Unless otherwise stated or obvious from the context, when used herein, the term "or" is to be understood to be inclusive.

[0065] When used to define compositions and methods, the term "comprising" means that the compositions and methods include the elements described, but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" means that the compositions and methods include the elements described and exclude other elements that are of any importance to the compositions and methods. For example, "consisting essentially of" refers to the administration of pharmacologically active agents that are explicitly described and excludes pharmacologically active agents that are not explicitly described. The term "consisting essentially of" does not exclude pharmacologically inactive or pharmacologically inert agents such as pharmaceutically acceptable excipients, carriers or diluents. When used to define compositions and methods, the term "consisting of" means excluding trace amounts of other ingredients and elements of substantial method steps. Embodiments defined by each of these transitional terms are within the scope of the present invention.

[0066] As used herein, the term "agonist" refers to a compound that combines with a receptor to produce a cellular response. An agonist can be a ligand that binds directly to the receptor. Alternatively, an agonist can combine with a receptor indirectly, for example by (a) forming a complex with another molecule that binds directly to the receptor, or (b) otherwise causing modification of another compound so that the other compound binds directly to the receptor.

[0067] As used herein, the term "antagonist" refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the effect of the agonist or inverse agonist on the receptor. However, an antagonist has no effect on constitutive receptor activity.

[0068] As used herein, the term "antibody" refers to a molecule that can bind to an epitope or antigenic determinant. The term is intended to include complete antibodies and antigen-binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fab, Fv, single-chain antibodies and single or multiple immunoglobulin variable chains or CDR domain designs, as well as bispecific and multispecific antibodies. Antibodies can come from any animal source. Preferably, the antibody is mammalian, such as humans, rodents, rabbits, goats, guinea pigs, camels, horses, etc. or other suitable animals. Antibodies can recognize polypeptide or polynucleotide antigens. The term includes active fragments, including, for example, antigen-binding fragments of immunoglobulins, variable and / or constant regions of heavy chains, variable and / or constant regions of light chains, complementary determining regions (CDRs) and framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered 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, miniantibodies, humanized antibody molecules, and any functional fragments derived from these molecules, wherein such fragments retain specific binding.

[0069] When used in this article, the term "antigen" is used in this article to refer to any substance that causes the immune system to produce antibodies or specific cell-mediated immune responses against it. Disease-related antigens are any substances related to any disease that cause the immune system to produce antibodies or specific cell-mediated immune responses against it. Antigens can be recognized by the immune system and / or can induce humoral immune responses and / or cause cellular immune responses of B and / or T lymphocyte activation. Antigens can have one or more epitopes (B and / or T cell epitopes). Antigens preferably react with their corresponding antibodies or TCRs in a highly selective manner, and do not react with numerous other antibodies or TCRs that may be evoked by other antigens. Antigens used herein can also be a mixture of several single antigens.

[0070] As used herein, the term "biologically active" entity or an entity with "biological activity" is an entity having the structure, regulation or biochemical function of a naturally occurring molecule or any function associated with or associated with a metabolic or physiological process. A biologically active polypeptide or fragment thereof includes a polypeptide or fragment thereof that can participate in a biological process or reaction and / or can produce a desired effect. The biological activity may include an increase in a desired activity or a decrease in an unwanted activity. For example, an entity exhibits biological activity when it participates in a molecular interaction with another molecule, when it has therapeutic value in alleviating a disease condition, when it has preventive value in inducing an immune response, or when it has diagnostic and / or prognostic value in determining the presence of a molecule. A biologically active protein or polypeptide may be naturally occurring, or it may be synthesized from known components, such as by recombinant or chemical synthesis, and may include heterologous components.

[0071] As used herein, the terms "cancer" and "carcinoma" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, epithelial cancers, lymphomas, sarcomas, blastomas, and leukemias. More specific examples of these cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, hepatoma, breast cancer, colon cancer, and head and neck cancer.

[0072] When used herein, the term "cell" refers to any prokaryotic, eukaryotic primary cell or immortalized cell line, any cell population in, for example, a tissue or organ. Preferably, the cell is of mammalian (eg, human) origin and can be infected by one or more pathogens.

[0073] As used herein, the term "co-administration" refers to the simultaneous presence of two agents in the blood. The two agents may be administered simultaneously or sequentially.

[0074] As used herein, the term "co-expressed" means that two different polypeptides are expressed simultaneously in a host cell, such that the two polypeptides can interact or bind and form a complex in the host cell or in the culture medium of the host cell.

[0075] As used herein, the term "disease" or "disorder" refers to a pathological condition, such as a pathological condition that can be identified as a deviation from a healthy or normal state by symptoms or other identifying factors. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative, inflammatory, immune, metabolic, infectious, and ischemic diseases.

[0076] 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 of ordinary skill in the art, the effective amount of a compound of the invention may vary with factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, and the patient.

[0077] 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 may be a direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of mRNA. Gene products also include RNA modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and glycosylation.

[0078] When used in this article, the term "host cell" refers to an individual cell or cell culture that can be or has been a recipient of any recombinant vector or isolated polynucleotide. The host cell can be a cell transfected, transformed, transduced or infected from any source including prokaryotic, eukaryotic, mammalian, avian, insect, plant or bacterial cells, or it can be a cell from any source of the nucleic acid described herein for propagation. The host cell includes the offspring of a single host cell, and the offspring may not necessarily be completely consistent with the original parent cell (in terms of morphology or a full set of DNA) due to natural, accidental or deliberate mutations and / or changes. The host cell includes cells transfected or infected with the recombinant vector or polynucleotide of the present invention in vivo or in vitro. The host cell comprising the recombinant vector of the present invention can be referred to as a "recombinant host cell".

[0079] Host cells include, but are not limited to, cells of mammals, plants, insects, fungi and bacteria. Bacterial cells include, but are not limited to, cells of species of gram-positive bacteria such as Bacillus, Streptomyces and Staphylococcus, and cells of gram-negative bacteria such as Escherichia and Pseudomonas. Fungal cells preferably include yeast cells such as Saccharomyces, Pichia pastoris and Hansenula polymorpha. Insect cells include, but are not limited to, cells of fruit flies and Sf9 cells. Plant cells include cells from crop plants such as cereals, medicinal and ornamental plants or bulbs, etc. Mammalian cells suitable for use in the present invention include epithelial cell lines (pig, etc.), osteosarcoma cell lines (human, etc.), neuroblastoma cell lines (human, etc.), epithelial cancers (human, etc.), glial cells (murine, etc.), liver cell lines (monkey, etc.), 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 cell line, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, NIH3T3 cells, 293T, REH and MCF-7 and hMSC cells.

[0080] When used in this article, the term "Fc" refers to a molecule or sequence comprising a sequence of a non-antigen binding fragment of a complete antibody, whether in monomeric or polymeric form. The original immunoglobulin source of native Fc is preferably of human origin, and can be any immunoglobulin (e.g., IgG1, IgG2). Native Fc consists of monomeric polypeptides, which can be connected into dimer or polymeric forms by covalent (i.e., disulfide bonds) and non-covalent bonding. The number of intermolecular disulfide bonds between the monomer subunits of native Fc molecules depends on class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2), in the range of 1 to 4.

[0081] As used herein, the term "Fc domain" or "Fc region" means the "crystallizable fragment" region of an immunoglobulin heavy chain. Typically, an Fc domain is capable of interacting with a second Fc domain to form a dimeric complex. The Fc domain may be capable of binding to cell surface receptors known as Fc receptors and / or proteins of the complement system, or may be modified to reduce or enhance these binding activities. The Fc domain may be derived from an IgG, IgA, IgD, IgM, or IgE antibody isotype and performs immunological activities, including opsonization, cell lysis, degranulation of mast cells, basophils, and eosinophils, and other Fc receptor-dependent processes, activation of the complement pathway, and in vivo protein stability.

[0082] "Fc domain" encompasses native Fc and Fc variant molecules and sequences as defined herein. Like Fc variants and native Fc, the term "Fc domain" includes molecules in monomeric or multimeric form, whether digested from intact antibodies or produced by recombinant gene expression or other means.

[0083] Fc fusion proteins have been reported to combine the Fc region of IgG with another protein such as domains of 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. Pat. Nos. 5,116,964 and 5,541,087).

[0084] The use of Fc fusions is known in the art (e.g., U.S. Pat. Nos. 7,754,855, 5,480,981, 5,808,029, WO7 / 23614, WO98 / 28427, and references cited therein). Fc fusion proteins may include variant Fc molecules (e.g., as described in U.S. Pat. No. 7,732,570). Fc fusion proteins may be dissolved in the cytoplasm or may be bound to the cell surface of cells with specific Fc receptors.

[0085] When used herein, the term "Fc variant" refers to a molecule or sequence that is modified from native Fc but still contains a binding site for the salvage receptor FcRn. International applications WO 97 / 34631 (published on September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and interactions with salvage receptors, and are incorporated herein by reference. Therefore, the term "Fc variant" includes molecules or sequences that are humanized from non-human native Fc. In addition, native Fc includes sites that can be removed due to providing structural features or biological activities that are not needed by the fusion molecule of the present invention. Thus, in certain embodiments, the term "Fc variant" encompasses molecules or sequences that lack one or more native Fc sites or residues that affect or participate in the following processes: (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity after expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than salvage receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in more detail below.

[0086] As used herein, the term "fusion protein" refers to a polypeptide comprising two or more regions from different or heterologous proteins that are covalently linked (i.e., "fused") by recombinant, chemical, or other suitable methods. If desired, the fusion molecule can be fused at one or more sites via peptide or other connector segments or sequences. For example, one or more peptide connectors can be used to assist in the construction of a fusion protein.

[0087] As used herein, the term "GC content" refers to the percentage of deoxyguanosine (G) and / or deoxycytidine (C) deoxyribonucleosides or guanosine (G) and / or cytidine (C) ribonucleoside residues that a nucleic acid sequence comprises.

[0088] As used herein, the term "high dose" means at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200% or even 300%) higher than the highest standard recommended dose of a particular compound for treating any human disease or condition.

[0089] As used herein, the term "immune response" refers to the process of stimulating immune cells and / or recruiting immune cells from the blood to lymphoid or non-lymphoid tissues through a multifactorial process involving different adhesion and / or activation steps. The activation condition leads to the release of cytokines, growth factors, chemokines and other factors, upregulating the expression of adhesion and other activation molecules on immune cells, promoting adhesion, morphological changes and / or extravasation accompanied by chemotaxis through tissues, increasing cell proliferation and cytotoxic activity, stimulating antigen presentation, and providing other phenotypic changes, including the generation of memory cell types. Immune response also means the activity of immune cells to inhibit or regulate the inflammatory or cytotoxic activity of other immune cells. Immune response refers to the activity of immune cells in vivo or in vitro.

[0090] In the context of two or more nucleic acid or polypeptide sequences, the term "identical" or "identity" percentage means that two or more sequences or subsequences are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity over a specified region (e.g., a region of an IL15 or IL15Rα sequence) when compared and aligned to achieve maximum correspondence over a comparison window or specified region) when measured using the BLAST or BLAST 2.0 sequence comparison algorithm using the default parameters described below or by manual alignment and visual inspection. The sequences are therefore said to be "substantially identical". This definition also refers to or can be applied to the complement of the test sequence. The definition also includes sequences with deletions and / or additions as well as sequences with substitutions. As described below, preferred algorithms can take gaps, etc. into account. Preferably, the identity exists over a region that is at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, typically over a region that is 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length or over the entire length of the amino acid or nucleic acid sequence.

[0091] For sequence comparison, usually a sequence serves as a reference sequence, and a test sequence is compared thereto. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used or optional parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence according to the program parameters.

[0092] A preferred example of an algorithm suitable for determining sequence identity and sequence similarity percentages is the BLAST algorithm, which is 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 through the National Center for Biotechnology Information at the World Wide Web site ncbi.nlm.nih.gov / . Default parameters or other non-default parameters may be used. As default parameters, the BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation value (E) of 10, M=5, N=-4 and a comparison of both chains. For amino acid sequences, the BLASTP program uses a wordlength (W) of 3, an expectation (E) of 10 and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) alignment (B) of 50, an expectation (E) of 10, M=5, N=-4 and a comparison of both strands as default parameters.

[0093] As used herein, the term "inhibition" refers to any measurable decrease in biological activity. Thus, as used herein, "inhibition" can be referred to as a percentage of normal activity levels.

[0094] As used herein, the term "interleukin 15" or "IL15" refers to a biologically active 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, meaning that the mutated protein ("mutant") has functionality similar to that of the native IL15 protein (75% or higher) in at least one functional assay. Functionally, IL15 is a cytokine that regulates the activation and proliferation of T cells and natural killer cells.

[0095] IL15 and IL2 share many biological activities, including binding to the IL2β / IL15β receptor subunit CD122. The number of CD8+ memory cells is controlled by the balance between IL15 and IL2. IL15 induces the activation of JAK kinases and the phosphorylation and activation of transcriptional activators STAT3, STAT5 and STAT6. IL15 also increases the expression of apoptosis inhibitors BCL2L1 / BCL-x(L), probably through the transcriptional activation activity of STAT6, thus preventing apoptosis. Two alternative splicing transcript variants of the IL15 gene encoding the same mature protein have been reported.

[0096] Exemplary functional assays for IL15 polypeptides include proliferation of T-cells (e.g. Montes et al., 2005 Clin Exp Immunol 142:292) and activation of NK cells, macrophages and neutrophils. Detection of the isolation and proliferation of specific immune cell subsets (i.e. 3 H-thymidine incorporation) are well known in the art. Cell-mediated cytotoxicity assays can be used to measure NK cell, macrophage and neutrophil activation. Cell-mediated cytotoxicity assays, including isotopes ( 51 Cr), dyes (e.g., tetrazolium salts, neutral red) or enzyme release are also well known in the art, and commercially available kits can be used (Oxford Biomedical Research, Oxford, M; Cambrex, Walkersville, Md.; Invitrogen, Carlsbad, Calif.). IL15 has also been shown to inhibit Fas-mediated apoptosis (e.g., Demirci et al., 2004 Cell Mol Immunol 1:123). Apoptosis assays, including, for example, TUNEL assays and Annexin V assays, are well known in the art, and commercially available kits can be used (R&D Systems, Minneapolis, Minn.) (e.g., Coliga et al., 1991-2006 Current Methods in Immunology John Wiley & Sons).

[0097] As used herein, the term "interleukin 15 receptor alpha" or "IL15Rα" refers to an interleukin 15 receptor alpha amino acid sequence from a mammalian species. Those skilled in the art will recognize that interleukin-15 receptor alpha nucleic acid and amino acid sequences are publicly available in gene databases such as GenBank, through the National Center for Biotechnology Information, and on the World Wide Web at ncbi.nlm.nih.gov. Exemplary native mammalian IL-15 receptor alpha nucleic acid or amino acid sequences can be from, for example, humans, primates, canines, felines, porcines, equines, bovines, sheep, rodents, murines, rats, hamsters, guinea pigs, and the like. Accession numbers for exemplary native mammalian IL-15 nucleic acid sequences include NM_172200.1 (human subtype 2) and NM_002189.2 (human subtype 1 precursor). Exemplary native mammalian IL-15 amino acid sequence accession numbers include NP_751950.1 (human isoform 2) and NP_002180.1 (human isoform 1 precursor).

[0098] As used herein, "interleukin 15 receptor alpha" or "IL15Rα" may also refer to a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the native mammalian IL15Rα amino acid sequence, having biological activity, and having functionality similar (75% or higher) to the native IL15Rα protein in at least one functional assay. IL15Rα is a cytokine receptor that specifically binds IL15 with high affinity. One functional assay is specific binding to the native IL15 protein.

[0099] As used herein, the term "isolated" molecule (e.g., polypeptide or polynucleotide) is a molecule that has been manipulated so as to exist at a higher concentration than in nature or has been removed from its native environment. For example, when at least 10% or 20% or 40% or 50% or 70% or 90% of the non-subject antibody material associated with the subject antibody in nature has been removed, the subject antibody is separated, purified, substantially separated or substantially purified. For example, a polynucleotide or polypeptide naturally present in a living animal is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated". In addition, for the purposes of the present invention, the recombinant DNA molecules contained in the vector are considered to be isolated. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules also include synthetically produced molecules. In addition, vector molecules contained in recombinant host cells are also isolated. Therefore, not all "isolated" molecules must be "purified".

[0100] As used herein, the term "linker" or "linker segment" refers to a molecule or group that connects two other molecules or groups. A peptide linker can allow the connected molecules or groups to obtain a functional configuration. The linker peptide preferably comprises 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 about 100 amino acids.

[0101] The components of fusion protein, such as cytokines or other bioactive molecules and any peptide connectors, can be organized in almost any way, as long as the fusion protein has the function it intends. Specifically, if necessary, each component of the fusion protein can be separated from another component by at least one suitable peptide connector segment or sequence. In addition, the fusion protein can include a label, such as to facilitate the modification, identification and / or purification of the fusion protein. More specific fusion proteins are in the embodiments described below.

[0102] As used herein, the term "low dose" refers to at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) lower than the lowest standard recommended dose of a particular compound formulated for a given route of administration for the treatment of any human disease or condition. For example, a low dose of a medicament formulated for administration by inhalation is distinguished from a low dose of the same medicament formulated for oral administration.

[0103] When used in this article, the term "medium" includes any culture medium, solution, solid, semisolid or rigid support, which can carry or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, Chinese hamster ovary syndrome (CHO) cells, prokaryotic host cells, Escherichia coli or Pseudomonas host cells and cell inclusions. Therefore, the term can encompass a culture medium in which the host cell has grown, such as a culture medium in which the polypeptide has been secreted, including a culture medium before or after the proliferation step. The term also encompasses a buffer or reagent containing a host cell lysate, such as when the polypeptide is produced intracellularly and the host cell is cracked or broken to release the polypeptide.

[0104] As used herein, the term "modulate" refers to directly or indirectly producing an increase or decrease, stimulation, inhibition, interference or blocking of a measured activity when compared to a suitable control. A "modulator" of a polypeptide or polynucleotide refers to a substance that affects, for example, increases, decreases, stimulation, inhibition, interference or blocking the measured activity of the polypeptide or polynucleotide when compared to a suitable control. For example, a "modulator" may bind with a measurable affinity and / or activate or inhibit a target, or directly or indirectly affect the normal regulation of receptor activity.

[0105] The term "operably linked" refers to a functional relationship 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. Operably linked nucleic acid sequences need not be physically adjacent to each other. The term "operably linked" also refers to a functional relationship between a nucleic acid expression control sequence (e.g., a promoter or an array of transcription factor binding sites) and a transcribable nucleic acid sequence, wherein the expression control sequence directs transcription of a nucleic acid corresponding to the transcribable sequence.

[0106] 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, vehicle, solvent or encapsulating material, which participates in carrying or transporting the subject agent 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 dosage form 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 sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; powered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; 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 glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffered saline; and other nontoxic compatible substances used in pharmaceutical dosage forms. Wetting agents, emulsifiers 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, flavoring and perfuming agents, preservatives and antioxidants, can also be present in the compositions.

[0107] 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 may include double-stranded, single-stranded or triple-helical sequences, and may include, but are not limited to, cDNA from viral, prokaryotic and eukaryotic sources, mRNA, genomic DNA sequences from viral (e.g., DNA viruses and retroviruses) or prokaryotic sources, RNAi, cRNA, antisense molecules, recombinant polynucleotides, ribozymes and synthetic DNA sequences. The terms also encompass sequences of any known base analogs including DNA and RNA. Nucleotides may be referred to by their commonly accepted single-letter codes.

[0108] Polynucleotides are not limited to polynucleotides occurring in nature, but also include polynucleotides in which non-natural nucleotide analogs and internucleotide linkages occur. Nucleic acid molecules may contain modified nucleic acid molecules (e.g., modified bases, sugars, and / or internucleotide linkages). Non-limiting examples of this type of non-natural structure include polynucleotides in which the sugar is not ribose, polynucleotides in which 3′-5′ and 2′-5′ phosphodiester bonds occur, polynucleotides in which reverse bonds (3′-3′ and 5′-5′) and branched structures occur. In addition, the polynucleotides of the present invention include non-natural internucleotide linkages such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), C1-C4 alkylphosphonate bonds of methylphosphonates, C1-C6 alkylphosphotriesters, phosphorothioates, and phosphorodithioates. In any case, the polynucleotides of the present invention maintain the ability to hybridize with target nucleic acids in a manner similar to natural polynucleotides.

[0109] Unless otherwise indicated or it is obvious from the context that this is not the case, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the explicitly indicated sequence. Degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced 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).

[0110] As used herein, the term "prevention" refers to a method for preventing, delaying, transferring or stopping the onset, occurrence, severity or recurrence of a disease or condition. For example, if there is a reduction or delay in the onset, occurrence, severity or recurrence of a disease or condition or one or more symptoms thereof in a subject susceptible to the disease or condition compared to a subject not receiving the method, the method is considered to be prevention. If there is a reduction or delay in the onset, occurrence, severity or recurrence of one or more symptoms of the disease or condition after receiving the method in a subject susceptible to the disease or condition compared to the development of the subject before receiving treatment, the disclosed method is also considered to be prevention. The reduction or delay in the onset, occurrence, severity or recurrence of osteoporosis can be a reduction of about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% or any amount therebetween.

[0111] Prevention and the like do not mean permanently preventing a subject from developing the particular disease or disorder. Prevention may require administration of multiple doses. Prevention may include preventing recurrence of the disease in a subject in which all disease symptoms have been resolved or preventing recurrence in a relapsing-remitting disease.

[0112] When used in this article, the term "promoter" refers to a DNA regulatory region that can bind to the RNA polymerase in mammalian cells and initiate the transcription of the downstream (3' direction) coding sequence that is operably connected thereto. The promoter sequence includes the minimum number of bases or elements necessary to initiate the transcription of the gene of interest at a detectable level higher than the background. A transcription initiation site and a protein binding domain (consensus sequence) responsible for the binding of the RNA polymerase may be present within the promoter sequence. Eukaryotic promoters usually, but not always, contain "TATA" boxes and "CAT" boxes. Promoters include promoters that are naturally adjacent to nucleic acid molecules and promoters that are not naturally adjacent to nucleic acid molecules. In addition, the term "promoter" includes inducible promoters, conditionally active promoters such as cre-lox promoters, constitutive promoters, and tissue-specific promoters.

[0113] When used in this article, the terms "protein" and "polypeptide" are used interchangeably and refer to polymers of amino acid residues, and are not limited to a minimum length. Therefore, peptides, oligopeptides, dimers, polymers, etc. are included in the definition. The definition covers both full-length proteins and fragments thereof. The term also includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, etc. In addition, a polypeptide may refer to a protein comprising modifications such as deletions, additions, and substitutions (usually conservative in nature) to the native sequence, as long as the protein maintains the desired activity. These modifications may be deliberate or may be accidental. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0114] As used herein, the term "purified" means that a protein may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e., native cells or host cells in the case of recombinantly produced proteins. Proteins that may be substantially free of cellular material include protein preparations having less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating proteins. When a protein or variant thereof is recombinantly produced by a host cell, the protein may be present in an amount of 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 cell. When the protein or its variant is produced by host cell recombinant production, the protein may be present in the culture medium at a dry weight concentration of 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. Thus, a "substantially purified" protein may have a purity level of at least about 80%, particularly a purity level of at least about 85%, more particularly a purity level of at least about 90%, a purity level of at least about 95%, a purity level of at least about 99% or more, when determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC and capillary electrophoresis.

[0115] The proteins and prodrugs of the invention are preferably isolated and / or purified after their preparation to obtain compositions containing an amount equal to or greater than 80% by weight ("substantially pure"), which are then used or formulated as described herein. In certain embodiments, the compounds of the invention are more than 95% pure.

[0116] 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. The ligand may belong to any class of biochemical or chemical compounds. The ligand is typically an extracellular molecule that, upon binding to a receptor, typically triggers a cellular response, such as initiating a signal transduction pathway. The receptor is not necessarily a membrane-bound protein.

[0117] As used herein, the term "recombinant" with respect to nucleic acid molecules means a polynucleotide of genomic, cDNA, viral, semisynthetic and / or synthetic origin that, by virtue of its origin or manipulation, is not accompanied by all or a portion of the polynucleotides with which it is associated in nature. The term "recombinant" with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" when applied to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

[0118] As used herein, the term "sample" refers to a sample or research sample from a human being, an animal, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid or condensate. The "sample" can be tested in vivo, such as without being taken out from the human being or animal, or it can be tested in vitro. The sample can be tested after processing, such as by a histological method. "Sample" also refers to, for example, cells that make up a fluid or tissue sample or cells separated from a fluid or tissue sample. "Sample" can also refer to cells, tissues, organs or fluids freshly obtained from a human being or an animal, or cells, tissues, organs or fluids that are processed or stored.

[0119] 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 a 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 is retained in an aqueous solution at a temperature above about 5-37° C., at a pH equal to or close to neutral, in the presence of low concentrations or in the absence of anionic or nonionic detergents. Under these conditions, a soluble protein typically has a sedimentation value, e.g., less than about 10 to 50 Svedberg units.

[0120] Aqueous solutions referred to herein typically have a buffer compound to establish a pH typically in the pH range of about 5-9 and an ionic strength range between about 2 mM and 500 mM. Protease inhibitors or mild nonionic detergents are sometimes added. In addition, carrier proteins (e.g., bovine serum albumin) may be added if desired. Exemplary aqueous buffers include standard phosphate buffered saline, tris buffered saline, or other well-known buffers and cell culture medium formulations.

[0121] As used herein, the term "soluble IL15 receptor alpha" refers to a form of IL15 receptor alpha that lacks the transmembrane anchoring portion of the receptor and is therefore capable of being secreted outside the cell rather than being anchored to the plasma membrane.

[0122] As used herein, the term "stimulate" refers to improving, amplifying, increasing, enhancing a physiological activity such as an immune response. Stimulation can be a positive change. For example, the increase can be 5%, 10%, 25%, 50%, 75% or even 90-100%. Other exemplary increases include 2 times, 5 times, 10 times, 20 times, 40 times or even 100 times.

[0123] As used herein, the terms "subject" and "patient" are used interchangeably herein and refer to living animals (human or non-human). The subject can be a mammal. The term "mammal" refers to any animal in the taxonomic classification class Mammalia. The mammal can be a human or a non-human mammal such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term "subject" does not exclude individuals who are completely normal or normal in all respects for a disease or condition.

[0124] As used herein, the term "inhibit" refers to reducing, weakening, decreasing, stopping or stabilizing a physiological activity such as an immune response. Inhibition can be a negative change. For example, the reduction can be 5%, 10%, 25%, 50%, 75% or even 90-100%. Exemplary reductions include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold or even 100-fold.

[0125] As used herein, the term "therapeutically effective amount" refers to a dose of one or more therapeutic agents sufficient to achieve the desired therapeutic effect with minimal or no undesirable side effects. The therapeutically effective amount can be readily determined by a skilled physician, for example, by first administering a low dose of the agent and then gradually increasing the dose until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0126] As used herein, the term "transfected" means having introduced DNA or RNA, with or without any accompanying facilitating agent such as liposome transfection reagent. Methods known in the art for transfection include, for example, calcium phosphate transfection, DEAE dextran transfection, protoplast fusion, electroporation and liposome transfection.

[0127] As used herein, the term "treating" a disease or disorder refers to mitigating, delaying or ameliorating such a condition or one or more symptoms of such a disease or condition before or after occurrence. Treatment can be directed to one or more effects or symptoms of a disease and / or underlying disease. Treatment can be any mitigation, and can be, but is not limited to, complete elimination of the disease or disease symptoms. When measured by any standard technique, this mitigation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% compared to an equivalent untreated control.

[0128] As used herein, the term "tumor" refers to any malignant or neoplastic cell.

[0129] When used in this article, the term "vector" refers to a nucleic acid molecule that can transfer genetic material to a host cell or organism. The vector can be composed of DNA or RNA. The vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases (which can be used to insert foreign DNA), and usually carries a selectable marker such as a gene encoding antibiotic resistance, and usually contains a recognition sequence (e.g., a promoter) for expressing the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0130] Any composition or method disclosed herein can be combined with one or more of any other compositions and methods provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0132] The present invention provides novel fusion proteins and therapeutic uses thereof. More specifically, the present invention provides novel IL15 fusion proteins and prodrugs thereof, compositions thereof, and methods for preparing the same, which can be used to treat a variety of different diseases and disorders such as hyperplasia, solid tumors, or hematopoietic malignancies and have reduced off-target toxicity and side effects during treatment.

[0133] In one aspect, the present invention generally relates to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of an interleukin 15 receptor (IL15R) or a fragment thereof; a second structural unit: an active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; and a first connector segment (L1), which covalently connects 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.

[0134] On the other hand, the present invention generally relates to a fusion protein. The fusion protein comprises: a first structural unit: a subunit of an interleukin 15 receptor (IL15R) or a fragment thereof; a second structural unit: an active IL15; a third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein; and a first connector segment (L1), which covalently connects the first, second and third structural units, wherein the second structural unit is at the N-terminus of the fusion protein, and the first structural unit is located between the second structural unit and the third structural unit.

[0135] In certain embodiments of the fusion protein, the subunit of IL15R is selected from the group consisting of an α subunit, a β subunit and a γ subunit.

[0136] In certain embodiments of the fusion protein, the subunit of IL15R is an α subunit.

[0137] In certain embodiments of the fusion protein, the fragment is the sushi domain of the α subunit of mouse IL15R, which has the amino acid sequence set forth in SEQ ID No.4.

[0138] In certain embodiments of the fusion protein, the fragment is the sushi domain of the α subunit of human IL15R, which has the amino acid sequence set forth in SEQ ID No.5.

[0139] In certain embodiments of the fusion protein, the IL15 is human or murine IL15.

[0140] In certain embodiments of the fusion protein, the IL15 is mouse IL15. In certain embodiments of the fusion protein, the mouse IL15 has the amino acid sequence set forth in SEQ ID No.1.

[0141] In certain embodiments of the fusion protein, the IL15 is human IL15. In certain embodiments of the fusion protein, the human IL15 has the amino acid sequence set forth in SEQ ID No.2.

[0142] In certain embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc fragment.

[0143] 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.

[0144] In certain embodiments of the fusion protein, the linker segment L1 comprises a plurality of GGGS.

[0145] In certain embodiments of the fusion protein, the linker segment L1 connecting the first structural unit to the third structural unit comprises the amino acid sequence set forth in SEQ ID No.9.

[0146] In certain embodiments of the fusion protein, the linker segment L1 connecting the first and second structural units comprises the amino acid sequence set forth in SEQ ID No.8.

[0147] In certain embodiments, the fusion protein further comprises: a fourth structural unit, which is located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor β subunit (Rβ); and a linker segment L2, which covalently connects the fourth structural unit and the remaining structural units of the fusion protein, wherein the first structural unit is covalently connected to the C-terminus of the fourth structural unit, and the second structural unit is located between the first structural unit and the third structural unit, and wherein the linker segment L2 can be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

[0148] In certain embodiments, the fusion protein further comprises: a fourth structural unit, which is located at the N-terminus of the fusion protein: the extracellular domain of the IL15 receptor β subunit (Rβ); and a linker segment L2, which covalently connects the fourth structural unit and the remaining structural units of the fusion protein, wherein the second structural unit is covalently connected to the C-terminus of the fourth structural unit, and the first structural unit is located between the second structural unit and the third structural unit, and wherein the linker segment L2 can be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

[0149] In certain embodiments, the amino acid sequence of mouse Rβ has the amino acid sequence set forth in SEQ ID No.6.

[0150] In certain embodiments, the amino acid sequence of human Rβ has the amino acid sequence set forth in SEQ ID No.7.

[0151] In certain embodiments of the fusion protein, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloproteinase.

[0152] In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 9 (MMP9).

[0153] In certain embodiments of the fusion protein, the matrix metalloproteinase is matrix metalloproteinase 14 (MMP14).

[0154] In certain embodiments of the fusion protein, the linker segment L2 comprises the amino acid sequence set forth in SEQ ID No. 10-23.

[0155] In another aspect, the present invention generally relates to a homodimeric or heterodimeric protein comprising a fusion protein disclosed herein.

[0156] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RA-IL15-Fc: a fusion protein of the sushi domain of the murine IL15 receptor α subunit, a linker segment L1, murine IL15, a linker segment L1, and human IgG1 Fc, and has an amino acid sequence such as that set forth in SEQ ID No.24.

[0157] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL15-RA-Fc: a fusion protein of the sushi domain of the human IL15 receptor α subunit, a linker segment L1, human IL15, a linker segment L1, and human IgG1 Fc, and has, for example, an amino acid sequence as set forth in SEQ ID No.25.

[0158] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL15-RA-Fc: a fusion protein of murine IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, and human IgG1 Fc, and has an amino acid sequence such as that set forth in SEQ ID No.26.

[0159] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of IL15-RA-Fc: a fusion protein of human IL15, linker segment L1, the sushi domain of the IL15 receptor α subunit, linker segment L1, and human IgG1 Fc, and has an amino acid sequence such as that set forth in SEQ ID No.27.

[0160] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: the extracellular domain of mouse IL15 receptor β subunit, linker segment L2, murine IL15, linker segment L1, the sushi domain of IL15 receptor α subunit, linker segment L1, a fusion protein of human IgG1 Fc, and has, for example, an amino acid sequence set forth in SEQ ID No.28.

[0161] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: the extracellular domain of human IL15 receptor β subunit, linker segment L2, human IL15, linker segment L1, the sushi domain of IL15 receptor α subunit, linker segment L1, a fusion protein of human IgG1 Fc, and has, for example, an amino acid sequence set forth in SEQ ID No.29-41.

[0162] In certain embodiments, the homodimeric or heterodimeric protein comprises a monomer of RB-IL15-RA-Fc: the extracellular domain of human IL15 receptor β subunit, linker segment L1, human IL15, linker segment L1, the sushi domain of IL15 receptor α subunit, linker segment L1, a fusion protein of human IgG1 Fc, and has, for example, an amino acid sequence set forth in SEQ ID No.42.

[0163] In certain embodiments, the homodimeric or heterodimeric protein is hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

[0164] In another aspect, the present invention generally relates to a substantially purified protein, such as a fusion protein or fragment disclosed herein.

[0165] In another aspect, the present invention generally relates to a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

[0166] In another aspect, the present invention generally relates to an expression vector comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

[0167] In another aspect, the present invention generally relates to a pharmaceutical composition comprising a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent.

[0168] In another aspect, the present invention generally relates to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent.

[0169] In another aspect, the present invention generally relates to a method for treating a disease or condition comprising administering to a patient in need thereof a therapeutically effective amount of a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein, wherein the disease or condition is selected from hyperplasia, solid tumors, or hematopoietic malignancies.

[0170] In certain embodiments, the disease or condition to be treated is hyperplasia.

[0171] In certain embodiments, the disease or condition to be treated is a solid tumor.

[0172] In certain embodiments, the disease or condition to be treated is a hematopoietic malignancy.

[0173] In certain embodiments, the subject to be treated is further administered one or more of chemotherapy, radiotherapy, targeted therapy, immunotherapy, or hormonal therapy.

[0174] In certain embodiments, the method further comprises administering to the subject a chemotherapeutic agent

[0175] In certain embodiments, the method further comprises administering radiation therapy to the subject.

[0176] In certain embodiments, the method further comprises administering to the subject a targeted therapy.

[0177] In certain embodiments, the method further comprises administering to the subject an immunotherapy.

[0178] In certain embodiments, the method further comprises administering hormone therapy to the subject.

[0179] As used herein, the term "chemotherapeutic agent" refers to a chemical compound that can be used to treat cancer. Examples of chemotherapeutic agents include erlotinib ( Genentech / OSI Pharm.), bortezomib ( Millennium Pharm.), Fulvestrant ( AstraZeneca), Sutent (SU11248, Pfizer), Letrozole ( Novartis), imatinib mesylate ( Novartis), PTK787 / ZK 222584(Novartis), oxaliplatin( Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, Wyeth), lapatinib ( GSK572016, Glaxo Smith Kline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs), and gefitinib ( AstraZeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents such as thiotepa and Cyclophosphamide, alkyl sulfonates such as busulfan, improsulfan and piposulfan, aziridines such as benzodopa, carboquinone, meturedopa and uredopa, ethyleneimines and methylmelamines including hexamethylmelamine, triethylenemelamine, triethylenephosphamide, triethylenethiophosphamide and trihydroxymethylmelamine, annona lactones (particularly bratacin and bratacinone), camptothecins (including the synthetic analog topotecan), bryostatin, callystatin, CC-1065 (including its synthetic analogs adolesin, carzelesin and biszelesin), nostoc (particularly nostoc 1 and nostoc 8), tail Aplysia, duocarmycin (including synthetic analogs KW-2189 and CB1-TM1), acanthopanax, cypermethrin, stoloniferin, sponge inhibin, nitrogen mustards such as chlorambucil, naphthyl mustard, chlorphosphamide, estramustine, ifosfamide, dichloromethyl diethylamine, dichloromethyl diethylamine oxide hydrochloride, melphalan, nembixin, phenylephrine, prednimustine, trofosfamide, uracil mustard, nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimustine, antibiotics such as enediyne antibiotics (e.g. calicheamicin, in particular calicheamicin gamma 11 and calicheamicin omega 11 (Angew Chem. Intl. Ed. Engl.(1994) 33:183-186), daunomycins including daunomycin A, bisphosphonates such as clodronate, esperamicin; and neocarcinogens and related chromogenic protein enediyne antibiotic chromophores), aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, actinomycin C, calabikine, carminomycin, carzinophilin, chromomycin, actinomycin D, daunomycin, detoxibacine, 6-diazo-5-oxo-L-norleucine , doxorubicin (doxorubicin, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolinyl-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, mexilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivetomycin, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin, streptomycin, streptozotocin, tuberculocin, ubenimex, zorubicin, antimetabolites such as methotrexate and 5-fluorouracil (5-FU), folic acid analogs such as dimethylfolate, methotrexate azathioprine, pterostilbene, trimetrexate, purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine, pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, deoxyfluridine, enocitabine, floxuridine, androgens such as dimethyltestosterone, drostanolone propionate, cyclothiodine, mestanolidine, testolactone, antiadrenal agents such as aminoglutethimide, mitotane, trilostane, folic acid supplements such as folinic acid, acetylglucosyl ester, aldophosphamide glycoside, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, Santren, edatrexate, defofamine, demeclofenac, diazocine, elfomithine, elliptonium acetate, epothilone, etoglucoside, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansines such as maytansine and ansamitocin, mitoxantrone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, podophyllic acid, 2-ethylhydrazide, procarbazine,. Polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane, rhizoxin, sizofiran, spirogermanium, tenuisporic acid, triazoquinone, 2,2',2"-trichlorotriethylamine, trichothecenes (particularly T-2 toxin, verracurin A, mycloracin A, and anguidine), urethane, vindesine, dacarbazine, mannitol mustard, dibromomannitol, dibromodulan, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxanes such as (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), (hydrogenated castor oil-free), albumin-engineered paclitaxel nanoparticle formulation (American Pharmaceutical Partners, Schaumberg, Illinois), and (docetaxel; Rhone-Poulenc Rorer, Antony, France), chlorambucil, (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, Vinorelbine, novantrone, teniposide, edatrexate, daunomycin, aminopterin, capecitabine Ibandronate, CPT-11, the topoisomerase inhibitor RFS 2000, difluoromethylornithine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids and derivatives of any of the foregoing agents.

[0180] Examples of the second (or additional) agent or therapy may include, but are not limited to, immunotherapy (e.g., PD-1 inhibitors (pembrolizumab, nivolumab, cemiplimab), PD-L1 inhibitors (atezolizumab, ovellumab, durvalumab), CTLA4 antagonists, cell signaling inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, temsirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab), Anti-cancer drugs (such as leukopenia, ranibizumab, pegaptanib, panitumumab, etc.), mitotic inhibitors (such as paclitaxel, vincristine, vinblastine, etc.), alkylating agents (such as cisplatin, cyclophosphamide, chlorambucil, carmustine, etc.), antimetabolites (such as methotrexate, 5-FU, etc.), intercalating anticancer agents (such as actinomycin, anthracycline, bleomycin, mitomycin-C, etc.), topoisomerase inhibitors (such as irinotecan, topotecan, teniposide, etc.), immunotherapy agents (such as interleukins, interferons, etc.) and anti-hormonal agents (such as tamoxifen, raloxifene, etc.).

[0181] In another aspect, the present invention generally relates to the use of a protein, such as a fusion protein or fragment disclosed herein, for treating or ameliorating a disease or disorder (eg, a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0182] In another aspect, the present invention generally relates to the use of polynucleotides encoding proteins, such as fusion proteins or fragments disclosed herein, for treating or ameliorating a disease or disorder (eg, hyperplasia, solid tumors, or hematopoietic malignancies).

[0183] In another aspect, the present invention generally relates to the use of a protein, such as a fusion protein or fragment disclosed herein, and a pharmaceutically acceptable excipient, carrier or diluent for the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., hyperplasia, solid tumors or hematopoietic malignancies).

[0184] In another aspect, the present invention generally relates to the use of polynucleotides encoding proteins, such as fusion proteins or fragments disclosed herein, and pharmaceutically acceptable excipients, carriers or diluents for the preparation of a medicament for treating or ameliorating a disease or disorder (e.g., hyperplasia, solid tumors or hematopoietic malignancies).

[0185] In certain embodiments, the drug is an anti-cancer drug.

[0186] 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.

[0187] In certain embodiments, the anti-cancer drug is effective against B-cell lymphoma or against colorectal cancer.

[0188] In another aspect, the present invention generally relates to a cell line comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

[0189] In another aspect, the present invention generally relates to a method for producing a protein, the method comprising culturing the cell line. In certain embodiments, the method further comprises purifying or isolating the produced protein, such as a fusion protein or fragment disclosed herein.

[0190] In another aspect, the present invention generally relates to a method for producing a protein. The method comprises: providing an expression vector encoding a protein, such as a fusion protein or fragment disclosed herein; introducing the expression vector into a host cell; culturing the host cell in a culture medium under conditions sufficient to express the protein; and purifying the protein from the host cell or culture medium.

[0191] Any suitable expression vector may be used. An exemplary expression vector is the pEE12.4 expression vector.

[0192] Any suitable host cells may be used, such as 293F and CHO cells.

[0193] Introduction of the expression vector may be achieved by any suitable transfection method, and may be via transient transfection or stable cell lines.

[0194] Any suitable purification method may be used. Exemplary purification methods are affinity chromatography by protein A / G or size exclusion methods.

[0195] In another aspect, the present invention generally relates to an isolated protein produced by the methods disclosed herein.

[0196] In certain embodiments, the isolated protein is substantially pure.

[0197] As disclosed herein, linker sequences can be used to link two or more of the biologically active polypeptides to produce a single chain molecule having a desired functional activity.

[0198] Any suitable connector can be adopted. Exemplary peptide connector sequences include peptide connector sequences with about 7 to 20 amino acids, for example about 8 to 16 amino acids. The connector sequence is preferably flexible, so that the biologically active polypeptide or effector molecule is not maintained under a single undesirable conformation. The connector sequence can be used for example to separate restriction sites from the fused molecule. Specifically, the peptide connector sequence can be placed to provide molecular flexibility. The connector preferably mainly comprises amino acids with small side chains such as glycine, alanine and serine, to provide flexibility.

[0199] Typically, the preparation of the fusion protein complex of the present invention can be achieved by the procedures disclosed herein and by recognized recombinant DNA technology, which involves, for example, polymerase chain amplification reaction (PCR), preparation of plasmid DNA, cutting DNA using restriction enzymes, preparation of oligonucleotides, connection of DNA, separation of mRNA, introduction of DNA into suitable cells, transformation or transfection of the host, and culture of the host. In addition, the fusion molecule can be separated and purified using chaotropic agents and well-known electrophoresis, centrifugation and chromatography methods. (For disclosures related to these methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York (1989)).

[0200] The present invention also provides nucleic acid sequences and DNA sequences encoding the fusion protein of the present invention. The DNA sequence can be carried by a vector suitable for extrachromosomal replication, such as a phage, virus, plasmid, phagemid, cosmid, YAC or episome. For example, a DNA vector encoding the desired fusion protein can be used to facilitate the preparation method described herein, and to obtain a significant amount of the fusion protein or its components. The DNA sequence can be inserted into a suitable expression vector, i.e., a vector containing the elements necessary for the transcription and translation of the inserted protein coding sequence. Various host-vector systems can be used to express the protein coding sequence. These systems can include mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.), insect cell systems infected with viruses (e.g., baculovirus), microorganisms containing yeast vectors, such as yeast, or bacteria transformed with phage DNA, plasmid DNA or cosmid DNA. Depending on the host-vector system utilized, any one of a large number of suitable transcription and translation elements can be used. (For disclosure 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)).

[0201] The fusion protein components encoded by the DNA vector can be provided in the format of a box. The term "box" means that each component can be easily replaced with another component by standard recombination methods. Specifically, when the encoded fusion complex is intended to be used to resist pathogens that may have or have the ability to develop serotypes, a DNA vector configured in the format of a box is particularly desirable.

[0202] In order to make a vector encoding a fusion protein complex, the sequence encoding the biologically active polypeptide is connected to the sequence encoding the effector peptide using a suitable ligase. The DNA encoding the proposed peptide can be obtained by isolating DNA from a natural source, for example, from a suitable cell line or by a known synthetic method, such as the phosphotriester method (Oligonucleotide Synthesis, IRL Press, MJ Gait, 1984). Synthetic oligonucleotides can also be prepared using a commercially available automatic oligonucleotide synthesizer. Once separated, the gene encoding the biologically active polypeptide can be amplified by PCR or other means known in the art. PCR primers suitable for amplifying the biologically active polypeptide gene can add restriction sites to the PCR product. The PCR product preferably includes a splicing site for the effector peptide and a leader sequence necessary for the correct expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably includes a sequence encoded for the connector sequence or a restriction enzyme site for connecting such a sequence.

[0203] The fusion protein described herein can be produced by standard recombinant DNA technology. For example, once the DNA molecule encoding the biologically active polypeptide is separated, the sequence can be connected to another DNA molecule encoding the effector polypeptide. The nucleotide sequence encoding the biologically active polypeptide can be directly linked to the DNA sequence encoding the effector peptide, or more generally, a suitable ligase can be used to insert the DNA sequence encoding the connector sequence discussed herein between the sequence encoding the biologically active polypeptide and the sequence encoding the effector peptide and connect. The obtained hybrid DNA molecule can be expressed in a suitable host cell to produce the fusion protein complex. The DNA molecules are connected to each other in a 5' to 3' direction so that after connection, the translation frame of the encoded polypeptide is not changed (i.e., the DNA molecules are connected to each other in the same frame). The obtained DNA molecule encodes the same frame fusion protein.

[0204] Other nucleotide sequences may also be included in the gene construct. For example, a promoter sequence that controls the expression of the sequence encoding the biologically active polypeptide fused to the effector peptide or a leader sequence that directs the fusion protein to the cell surface or culture medium may be included in the construct or present in the expression vector into which the construct is inserted.

[0205] In obtaining variant biologically active polypeptide IL15, IL15R or Fc domain encoding sequences, 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. Specifically, it is well known that conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid of similar size, charge, polarity and conformation, are unlikely to significantly alter protein function. The 20 standard amino acids that are components of proteins can be broadly classified into four groups of conservative amino acids as follows: the non-polar (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 contains aspartic acid and glutamic acid. The replacement of one amino acid in a protein by another amino acid in the same group is unlikely to have an adverse effect on the biological activity of the protein. In other cases, modifications to amino acid positions can be made to reduce or increase the biological activity of the protein. Such changes can be introduced randomly or by site-specific mutagenesis based on the known or inferred structural or functional properties of the target residue. After the variant protein is expressed, changes in biological activity caused by the modifications can be easily assessed using binding or functional assays.

[0206] Homology between nucleotide sequences can be determined by DNA hybridization analysis, where the stability of double-stranded DNA hybrids depends on the degree of base pairing that occurs. Conditions of high temperature and / or low salt content reduce the stability of the hybrids and can be altered to prevent annealing of sequences with less than a selected degree of homology. For example, for sequences with about 55% GC content, 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, 50-65° C., 1×SSC and 0.1% SDS indicate about 82-97% specificity, and 52° C., 0.1×SSC and 0.1% SDS indicate about 99-100% specificity. A large number of computer programs for comparing nucleotide and amino acid sequences (and measuring homology degrees) are also available. Readily available algorithms for sequence comparison and multiple sequence alignment are the Basic Local Alignment Search Tool (BLAST) and the ClustalW programs, respectively.

[0207] A large number of strategies can be used to express the protein fusion complex of the present invention. For example, the above-mentioned fusion protein construct can be incorporated into a suitable vector by known means, such as using restriction enzymes to make an incision in the vector for inserting the construct, and then connected. The vector containing the gene construct is then introduced into a suitable host for expressing the fusion protein (for disclosures related to these methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989)).

[0208] The selection of suitable vectors can be made empirically based on various factors related to the cloning scheme. For example, the vector should be compatible with the host used and have a suitable replicon for the host. In addition, the vector must be able to accommodate the DNA sequence encoding the fusion protein complex to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably those that can be easily transformed and show rapid growth in culture medium. Specifically, preferred host cells include prokaryotes such as Escherichia coli, Bacillus subtilis, etc. and eukaryotic organisms such as animal cells and yeast strains such as Saccharomyces cerevisiae. Mammalian cells are generally preferred, particularly J558, NSO, SP2-0 or CHO. Other suitable hosts include, for example, insect cells such as Sf9. Conventional culture conditions are used. Referring to Sambrook, supra. Stable transformed or transfected cell lines can then be selected. Cells expressing the fusion protein complex of the present invention can be determined by known procedures. For example, the expression of the fusion protein complex connected to an immunoglobulin can be determined by ELISA specific for the immunoglobulin connected and / or by immunoblotting. Other methods for detecting the expression of fusion proteins comprising a biologically active polypeptide linked to an IL12 or IL12R domain are disclosed in the Examples.

[0209] Host cells can be used for preparation purposes to breed nucleic acids encoding desired fusion proteins or their components. Host cells can include prokaryotic or eukaryotic cells specifically intended to produce the fusion proteins therein. Therefore, host cells specifically include yeast, flies, worms, plants, frogs, mammalian cells and organs capable of breeding nucleic acids encoding the 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).

[0210] Host cells capable of propagating nucleic acids encoding the desired fusion protein complex also encompass non-mammalian eukaryotic cells, including insects (e.g., S. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha; as generally reviewed by Fleer, R., 1992 Current Opinion in Biotechnology, 3(5): 486496), fungi, and plant cells. Certain prokaryotes such as Escherichia coli and Bacillus are also contemplated.

[0211] The nucleic acid encoding the desired fusion protein can be introduced into the host cell by standard techniques for transfecting cells. The term "transfection" is intended to encompass all conventional techniques for introducing nucleic acid into a host cell, including calcium phosphate coprecipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, viral transduction and / or integration.

[0212] According to the present invention, various different promoters (transcription initiation regulatory regions) can be used. The selection of a suitable promoter depends on the proposed expression host. Promoters from heterologous sources can be used as long as they are functional in the selected host.

[0213] Promoter selection also depends on the required efficiency and level of peptide or protein production. In order to sharply improve the protein expression level in Escherichia coli, inducible promoters such as tac are usually used. Protein overexpression may be harmful to host cells. Therefore, host cell growth may be limited. The use of inducible promoter systems allows the host cells to be cultured to an acceptable density before the induction of gene expression, thereby contributing to higher product yields.

[0214] According to the present invention, a variety of signal sequences can be used. Signal sequences homologous to the biologically active polypeptide coding sequence can be used. Alternatively, signal sequences selected or designed for efficient secretion and processing in the expression host can 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 can be linked through a short nucleotide bridge.

[0215] The expression construct can be assembled using known recombinant DNA techniques. Restriction enzyme digestion and ligation are basic steps for joining two DNA fragments. Polylinkers and conjugates can be used to help join selected fragments. The expression construct can usually be assembled in a stage utilizing multiple rounds of restriction, ligation, and transformation of E. coli. A large number of cloning vectors suitable for the construction of expression constructs are known in the art (λZAP and pBLUESCRIPT SK-1, Stratagene, La Jolla, Calif.; pET, Novagen Inc., Madison, Wis.; pEE12.4, Lonza Biologics, Basel, Switzerland).

[0216] The expression construct can be transformed into the host along with the linear or circular cloning vector construct, or can be removed from the cloning vector and used as is or introduced into a delivery vector. The 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 known gene transfer system (e.g., natural competence, chemical-mediated transformation, protoplast transformation, electroporation, bioprojectile transformation, transfection or conjugation). The selected gene transfer system depends on the host cell and vector system used.

[0217] The present invention also provides a production method for separating the fusion protein of interest. In the method, a host cell (e.g., yeast, fungi, insect, bacteria or animal cell) into which a nucleic acid encoding the protein of interest that is operably connected to a regulatory sequence is introduced is grown in a culture medium on a production scale to stimulate the transcription of the nucleotide sequence encoding the fusion protein of interest. Subsequently, the fusion protein of interest is separated from the host cell of the harvest or from the culture medium. Standard protein purification techniques can be used to separate the protein of interest from the cells of the culture medium or the harvest. Specifically, the purification technique can be used to express and purify the desired fusion protein on a large scale (i.e., with at least milligrams) from various devices, including roller bottles, rotary shake bottles, tissue culture plates, bioreactors or fermenters.

[0218] The protein fusion complex expressed can be separated and purified by known methods. Usually, the culture medium is centrifuged or filtered, and then the supernatant is purified by affinity or immunoaffinity chromatography, such as protein A or protein G affinity chromatography or includes the use of the immunoaffinity scheme of the monoclonal antibody of the TCR or its immunoglobulin region connected in conjunction with the fusion complex expressed. The fusion protein of the present invention can be separated and purified by the appropriate combination of known technology. These methods include, for example, salt precipitation and solvent precipitation using methods such as solubility, methods such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis using methods such as dialysis, ultrafiltration, gel filtration and SDS-polyacrylamide gel electrophoresis using methods such as ion exchange column chromatography using charge difference, methods such as affinity chromatography using specific affinity, methods such as reversed-phase high performance liquid chromatography using methods such as hydrophobicity difference, and methods such as isoelectric focusing electrophoresis using isoelectric point difference, metal affinity columns such as Ni-NTA. (For disclosure 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)).

[0219] Preferably, the fusion protein of the present invention is substantially pure. That is, the fusion protein has been separated from the cellular components that naturally accompany it, so that the fusion protein is preferably present with at least 80% or 90% to 95% homogeneity (w / w). For many pharmaceutical, clinical and research applications, fusion proteins with at least 98 to 99% homogeneity (w / w) are most preferred. Once substantially purified, the fusion protein should be substantially free of contaminants for therapeutic applications. Once partially purified or purified to significant purity, the soluble fusion protein can be used therapeutically or for performing in vitro or in vivo assays disclosed herein. Significant purity can be determined by various standard techniques such as chromatography and gel electrophoresis.

[0220] The present invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of the composition, fusion protein, polynucleotide, gene construct, vector or host cell according to the present invention and a pharmaceutically acceptable excipient or medium.

[0221] Preferred excipients for the present invention include carbohydrates, starch, cellulose, gums and proteins. In a preferred embodiment, the pharmaceutical composition of the present invention is formulated into a pharmaceutical form for administration as a solid (e.g., tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids that can be reconstructed to provide a liquid form, etc.), a liquid (e.g., a solution, a suspension, an emulsion, an elixir, a lotion, an oil, etc.) or a semisolid (gel, ointment, cream, etc.). The pharmaceutical composition of the present invention can be administered by any route, including but not limited to oral, intravenous, intramuscular, intraarterial, intramedullary, intracapsular, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual or rectal routes. The different administration forms of active pharmaceutical ingredients, excipients to be used and the revisions of their manufacturing procedures can be found in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000). Examples of pharmaceutically acceptable media are known in the state of the art and include phosphate-buffered saline solutions, water, emulsions such as oil / water emulsions, different types of wetting agents, sterile solutions, etc. Compositions comprising said media can be formulated by conventional procedures known in the state of the art.

[0222] In the case where the pharmaceutical composition of the present invention comprises nucleic acid (polynucleotide, vector or gene construct of the present invention), the present invention contemplates a pharmaceutical composition specially prepared for administration of the nucleic acid. The pharmaceutical composition may comprise the nucleic acid in naked form, in other words, there is no compound protecting the nucleic acid from being degraded by the nuclease of the organism, which has the advantage of eliminating the toxicity associated with the reagent for transfection. For the naked compound, suitable routes of administration include intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucosal, topical and oral routes (Templeton, 2002DNA Cell Biol., 21: 857-867). Alternatively, the nucleic acid can be administered as part of a liposome, coupled to cholesterol or coupled to a compound capable of promoting translocation through a cell membrane, such as the Tat peptide derived from the TAT protein of HIV-1, 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 described, for example, in WO07069090 (Lindgren et al., 2000 Trends Pharmacol. Sci 21:99-103; Schwarze et al., 2000 Trends Pharmacol. Sci. 21:45-48; Lundberg et al., 2003 Mol. Therapy 8:143-150; and Snyder et al., 2004 Pharm. Res. 21:389-393). Alternatively, the polynucleotide may form part of a plasmid vector or a viral vector, preferably an adenovirus-based vector, in an adeno-associated virus or in a retrovirus, such as a virus based on murine leukemia virus (MLV) or administered on a lentivirus (HIV, FIV, EIAV).

[0223] The compositions of the present invention may be administered at less than 10 mg / kg 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 / kg body weight, and less than 200 nmol of the agent, in other words about 4.4×10 16The bifunctional polynucleotides and compositions of the present invention can be administered directly to an organ in which the target mRNA is expressed, in which case between 0.00001 mg and 3 mg / organ will be administered, or preferably between 0.0001 and 0.001 mg / organ, about 0.03 to 3.0 mg / organ, about 0.1 to 3.0 mg / organ, or between 0.3 to 3.0 mg / organ.

[0224] The administration will depend on the severity of the condition to be treated and the reaction to the condition, and can vary between a few days to a few months or until the condition is observed to be relieved. The most suitable administration can be determined by regularly measuring the concentration of the agent in the patient's organism. The most suitable administration can be determined from the EC50 value obtained by in vitro or in vivo testing in animal models in advance. The unit dose can be once a day or less than once a day, preferably less than once every 2,4,8 or 30 days. Alternatively, an initial dose can be administered, followed by one or several maintenance doses of an amount generally lower than the initial dose. The maintenance regimen can include treating the patient with a dosage ranging from 0.01 μg to 1.4 mg / kg body weight / day, such as 1, 0.1, 0.01, 0.001 or 0.00001 mg / kg body weight / day. The maintenance dose is preferably administered once every 5, 10 or 30 days at most. The treatment must continue for a period of time, and the time varies according to the type of change experienced by the patient, its severity and the patient's condition. After treatment, the patient's evolution must be monitored in order to determine whether the dose should be increased if the disease is not responsive to the treatment, or decreased if improvement of the disease or unwanted secondary effects are observed.

[0225] The daily dose may be administered in a single administration or in two or more administrations, depending on the circumstances. If repeated administration or frequent administration is required, implantation of an administration device such as a pump, a semi-permanent catheter (intravenous, intraperitoneal, intracisternal or intracapsular) or a reservoir may be recommended.

[0226] The compositions of the present invention are administered according to methods known to experts in the art, including but not limited to intravenous, oral, nasal, parenteral, topical, transdermal, rectal administration, etc.

[0227] The following examples are intended to illustrate the practice of the present invention but are not intended to limit it in any way. Example

[0228] The following examples describe certain exemplary embodiments of compounds prepared according to the invention of the present disclosure. It should be recognized that the general methods described below, and other methods known to those of ordinary skill in the art, can be applied to the compounds and subclasses and species disclosed herein.

[0229] Example 1. Construction of fusion protein

[0230] A. Construction of four fusion proteins

[0231] Construction of control protein: IL15-Fc

[0232] Mouse IL15 was fused to the N-terminus of hIgG Fc (named IL15-Fc). Figure 1 A. The amino acid sequence of mouse IL15 is SEQ ID No. 1. The amino acid sequence of hIgG Fc is SEQ ID No. 3.

[0233] Two formats of Super IL15:

[0234] Schematic diagrams of IL15Rαsushi-IL15-Fc (named RA-IL15-Fc) and IL15-IL15Rαsushi-Fc (named IL15-RA-Fc) are shown. Figure 1 B and Figure 1 C. Similar in biological activity, the two structures are interchangeably referred to as super IL1. The amino acid sequence of mouse IL15Rαsushi is SEQ ID No.4. The amino acid sequence of human IL15Rαsushi is SEQ ID No.5. The sequence of the linker between IL15Rαsushi and IL15 is SEQ ID No.8. Mouse RA-IL15-Fc and IL15-RA-Fc are fully represented by SEQ ID No.24 and SEQ ID No.26. Human RA-IL15-Fc and IL15-RA-Fc are fully represented by SEQ ID No.25 and SEQ ID No.27.

[0235] Prodrugs:

[0236] The ECD (extracellular domain) of IL15Rβ was fused to the N-terminus of IL15-RA-Fc via the linker segment L2.

[0237] A schematic diagram of IL15RβECD-L2-IL15-IL15Rαsushi-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 of MMP9 or MMP14. The amino acid sequence of the MMP9 substrate linker is SEQ ID No. 10. The amino acid sequence of the MMP14 substrate linker is SEQ ID Nos. 11-23.

[0238] B. Construction, transfection, expression and purification of fusion proteins

[0239] 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 agarose gel. All proteins were quantified by ELISA and SDS-PAGE.

[0240] The detailed process is described as follows.

[0241] Construction of fusion proteins

[0242] IL15, IL15RA and IL15RB ECDs were synthesized and cloned into pEE12.4-IgGκ-hIgG1 Fc plasmid containing mouse IgGκ leader sequence and human IgG1 Fc. Plasmids were extracted using a standard commercial plasmid extraction kit and stored at -80°C.

[0243] Transfection of fusion proteins

[0244] 293F cells using CD OptiCHO TM The cells were cultured in the culture medium and incubated in a 37°C, 8% CO2 incubator with shaking at 135 rpm. Two days before transfection, the cells were plated at 0.6-0.8×10 6 Cells / mL were plated at a density of approximately 2.5-3.5×10 6 Collect cells at a density of 10 cells / mL, wash with Freestyle 293 medium, and then resuspend in 200mL Freestyle 293. Dilute DNA (600μg) with 5mL Freestyle 293 and filter through a 0.22μm filter. Dilute PEI (1.8mg) with 5mL Freestyle 293 and filter through a 0.22μm filter. Mix DNA and PEI and incubate at room temperature for 5min, then mix with cells in a shake flask. Place the shake flask in a 37°C, 8% CO2 incubator and shake at 85rpm. Add 200mL EX-CELL 4 hours after transfection. TM293 medium, shaking at 135 rpm. 20 hours after transfection, add 3.8 mM VPA. Collect supernatants on days 4 to 7 after transfection, when cell viability is above 70%.

[0245] Purification of fusion proteins

[0246] The fusion protein was purified using a protein A-Sepharose column according to the manual (Repligen Corporation).

[0247] Binding buffer: 20 mM sodium phosphate, pH 7.0

[0248] Elution buffer: 0.1 M glycine, pH 2.7

[0249] Regeneration buffer: 1M NaOH

[0250] Neutralization buffer: 1M Tris-HCl, pH 9.0

[0251] All buffers were filtered through a 0.45 μm filter.

[0252] (1) The sample was centrifuged at 8000×rpm for 2 hr to remove cells and then filtered through a 0.45 μm filter. NaN3 was added to a final concentration of 0.05% to prevent bacterial growth.

[0253] (2) If the column was preserved with 20% ethanol, wash it with 5 column volumes of distilled water at a linear flow rate of 50 to 100 cm / h.

[0254] (3) Wash the column with 5 to 10 column volumes of elution buffer to wash away impurities.

[0255] (4) The column was equilibrated with 5 to 10 column volumes of binding buffer at a linear flow rate of 50 to 100 cm / hr.

[0256] (5) Apply the pretreated sample to the column.

[0257] (6) Wash the column with 5 to 10 column volumes of binding buffer.

[0258] (7) Elute the column in a 1.5 mL elution tube.

[0259] The results of SDS-PAGE electrophoresis of the purified fusion protein are shown in Figure 2 In the figure, lane S1 was loaded with IL15-Fc, lane S2 was loaded with super IL15, and lane S3 was loaded with RB-IL15-RA-Fc.

[0260] Example 2. Biological functions of super IL15 fusion protein

[0261] A. Promote lymphocyte proliferation

[0262] We first characterized the ability of interleukin 15 (IL-15) to stimulate proliferation of the murine T cell line CTLL-2. In this protocol, CTLL-2 cells were cultured in the presence of serial dilutions of murine IL-15 and their growth was measured by CCK8.

[0263] Use the following procedure:

[0264] (1) CTLL2 cells were cultured using CTLL-2 assay medium supplemented with 100 U / mL recombinant human IL-2.

[0265] (2) Collect CTLL-2 cells in logarithmic phase growth 24 to 48 h after subculturing and wash them twice to remove residual IL-2. Resuspend the cells in 5 to 10 mL of CTLL-2 assay medium, count the cells, and adjust the concentration to 2 × 10 4 cells / mL.

[0266] (3) Dilute the sample using CTLL-2 assay medium. The initial maximum concentration is 10 μg / mL. Perform 1:10 serial dilutions in 7 tubes.

[0267] (4) Add 100 μL of cell suspension (2×10 3 cells / well); add 100 μL of sample to each well. Include a row of wells containing only 200 μL of assay medium as a negative control.

[0268] (5) Cover the plate and incubate for 48 to 72 hrs.

[0269] (6) Add 20 μL CCK8. After 2 to 4 hrs, read the OD450 and OD630 of each well using a microtiter plate reader.

[0270] The results are shown in Figure 3 , which showed that (1) the biological activities of the two forms of super IL15 are similar, that is, in the fusion protein, whether the IL15 fragment or the IL15Rαsushi fragment comes first, the function of super IL15 is not affected; and (2) compared with IL15-Fc, super IL15 has a biological activity that is increased by about 100 times.

[0271] B. The fusion fragment of IL15RβECD can block the biological function of super IL15

[0272] The proliferative capacity of murine RB-IL15-RA-Fc and super IL15 against CTLL2 was examined by CCK8 assay. Figure 4 The results shown in show that the biological activity of RB-IL15-RA-Fc was reduced 100-fold. This suggests that the extracellular domain of IL-15Rβ can block the biological function of super IL-15.

[0273] C. Antitumor effects and systemic toxicity in different tumor models

[0274] A20 Model

[0275] Experiment 1 (25 μg): A20 cells (3×10 6 ) was injected subcutaneously into the right flank of Balb / c mice. 3 ) were treated with 25 μg Super IL15 intratumorally (it) and intravenously (iv). The control group was treated with PBS. Tumor volume = length × width × height / 2. Tumor growth curve was recorded.

[0276] Results: (1) In the intratumoral treatment group, tumors from all mice showed complete regression ( Figure 5A Mice that had experienced complete tumor regression were re-challenged with a lethal dose of A20 cells. All mice rejected the re-challenged tumors, indicating a strong memory response ( Figure 5C ). (2) All mice died after the second dose of iv treatment, indicating severe systemic toxicity ( Figure 5B ).

[0277] Experiment 2 (12.5 μg): A20 cells (3×10 6 ) was injected subcutaneously into the right flank of Balb / c mice. 3 ) were treated with 12.5 μg Super IL15 intratumorally (it) and intravenously (iv). The control group was treated with PBS. Tumor volume was defined as length × width × height / 2. Tumor growth curves were recorded.

[0278] result( Figure 7 ): In the intratumoral administration treatment group, 100% of the mice achieved complete regression. In contrast, 20% of the mice treated intravenously achieved complete regression, and the tumors of the remaining mice were partially controlled.

[0279] MC38 Model

[0280] Experiment: MC38 cells (5×10 5) was injected subcutaneously into the right flank of C57 mice. 3 ) were treated with 25 μg Super IL15 intratumorally (it) and intravenously (iv). The control group was treated with PBS. Tumor volume = length × width × height / 2. Tumor growth curve was recorded.

[0281] Results: In the intratumoral administration treatment group, 50% of the mice achieved complete regression ( Fig. 6A ), and the survival rate was significantly improved ( Figure 6B In contrast, mice given intravenous administration did not achieve complete tumor regression ( Fig. 6A ), and the survival rate of mice was slightly improved ( Figure 6B ).

[0282] The above results suggest that super IL15 appears to act locally in the tumor microenvironment (TME).

[0283] Example 3. Comparison of tumor therapeutic effects and side effects between super IL15 and RB-IL15-RA-Fc

[0284] A20 cells (3×10 6 ) was injected subcutaneously into the right flank of Balb / c mice. 3 ) were treated with 12.5 μg super IL15 or Rβ-IL15-RA-Fc intraperitoneally (ip). Tumor growth was measured twice a week. Serum was collected 20 hr after the second injection. Cytokine levels in serum were measured by cytometric bead array (CBA) and tumor curves were recorded.

[0285] The following CBA regimen was used:

[0286] (1) Serum was collected from the ophthalmic vein and stored at -80°C.

[0287] (2) CBA kits from BD were used to evaluate IL12p70, IL-6, IFN-γ, TNFα, MCP1, and IL-10 in serum.

[0288] (3) The standards were reconstituted with 2.0 mL of assay diluent and then recalibrated at room temperature for at least 15 min. The standards were serially diluted at the following ratios: 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, 1:128, and 1:256.

[0289] (4) Mix the Th1 / Th2 / Th17 cytokine capture beads. Determine the number of assay tubes (including standards and controls) required for the experiment. Vortex each capture bead suspension vigorously for 3 to 5 seconds before mixing. For each assay tube to be analyzed, add a 2 μL aliquot of each capture bead to a single tube. Add a 10 μL aliquot of mouse Th1 / Th2 / Th17PE detection reagent to the tube and vortex thoroughly.

[0290] (5) Perform Th1 / Th2 / Th17 cytokine assay: Vortex the mixed capture beads and add 20 μL to all assay tubes. Add 50 μL of mouse Th1 / Th2 / Th17 cytokine standard dilution to the control tube. Add 50 μL of each unknown sample to the sample assay tube with the appropriate label. Incubate the assay tubes at room temperature in the dark for 2 hr.

[0291] (6) Add 1 mL of wash buffer to each assay tube and centrifuge at 300 g for 5 min.

[0292] (7) Carefully aspirate the supernatant from each assay tube and discard. Add 300 μL of wash buffer to each assay tube to resuspend the bead pellet.

[0293] (8) Samples were analyzed by flow cytometry and cytokine levels were calculated based on standards.

[0294] The therapeutic effect is shown in Fig. 8A In the present study, it was shown that the therapeutic effect of intravenously administered RB-IL15-RA-Fc was similar to that of super IL15.

[0295] The comparison results of serum inflammatory factor levels are shown in Figure 8B The results showed that the toxic side effects of RB-IL15-RA-Fc were significantly reduced compared with super IL15.

[0296] A20 cells (3×10 6 ) was injected subcutaneously into the right flank of Balb / c mice. 3 Mice with 25 μg of super IL15 or RB-IL15-RA-Fc were treated intraperitoneally (ip). Tumor growth was measured twice a week. Serum was collected 20 hr after the second injection. Cytokine levels in serum were measured by cytometric bead array (CBA) and tumor curves were recorded.

[0297] Results: After tumor re-challenge and treatment with super IL15, the tumor-bearing mice became significantly ill with severe weight loss, decreased mobility, ragged hair, and all died within one day after the second treatment. In contrast, none of the mice treated with RB-IL15-RA-Fc died, and no mouse appeared unhealthy. The survival curve for RB-IL15-RA-Fc was significantly longer than that for super IL15. The survival curves are shown in Figure 2. Fig. 9A In the Fig. 9B middle.

[0298] Taken together, RB-IL15-RA-Fc reduces the toxic side effects of super IL15.

[0299] Analogous human versions of various IL15 fusion proteins and prodrugs were also generated and tested in vitro. Human protein production followed the cloning, transfection, and purification protocols described above for murine protein production.

[0300] Recombinant human MMP-14 / MT1-MMP (R&D Systems) was activated and incubated with IL15 fusion protein at 37°C for 24 hrs to confirm prodrug activation and cleavage at the L2 linker site.

[0301] The results of SDS-PAGE electrophoresis of the purified human fusion protein incubated with or without MMP14 at 37°C for 24 hrs are shown in Fig.10 middle.

[0302] Functionality of human RB-IL15-RA-Fc using HEK-Blue TM IL-2 reporter cell assay (Invivogen). After IL-2 stimulation, HEK-Blue TM IL-2 cells initiate STAT5 activation and subsequent SEAP secretion. STAT5-induced SEAP levels can be measured using QUANTI-Blue TM Since IL15 binds to and signals through a complex consisting of the IL-2 / IL-15 receptor β chain and common γ chain, HEK-Blue TM IL-2 cell lines can also be used to measure the functional activity of IL15 and / or pro-IL15.

[0303] Use the following HEK-Blue IL-2 reporter cell assay:

[0304] (1) HEK-Blue IL-2 cells were gently rinsed in PBS and cultured at 1 x 10 6Cells were suspended at a density of 10 cells / mL in fresh pre-warmed assay medium (DMEM, 4.5 g / L glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated PBS) at 56°C for 30 mins.

[0305] (2) The samples were serially diluted in a flat-bottom 96-well plate and incubated with 50 μL of cell suspension (~50,000 cells) per well at 37°C in a CO2 incubator for 20-24 hrs.

[0306] (3) In each well of a flat-bottom 96-well plate, 20 μL of induced HEK-Blue IL-2 cell supernatant was mixed with 100 μL of resuspended QUANTI-Blue TM The solution was incubated in a 37°C incubator for 15-min to 1 hr.

[0307] (4) SEAP levels were determined using a spectrophotometer at 650 nm.

[0308] result: Fig.11 It was confirmed that RB-L2-15RA-Fc, which was constructed to have an MMP14 substrate sequence embedded in the linker segment (L2) and incubated with MMP14, exhibited the same level of function as 15RA-Fc with or without MMP14 incubation. Consistently, RB-L1-15RA-Fc, which was constructed to have no MMP14 substrate sequence embedded in the linker segment (L1), performed similarly to the sample without MMP14 incubation. The construct symbol 15RA is an abbreviation of IL15-L1-RA.

[0309] Sequence Listing

[0310] SEQ ID No. 1: Mouse IL15

[0311] NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTS

[0312] SEQ ID No. 2: Human IL15

[0313] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS

[0314] SEQ ID No.3: Human IgG1-Fc

[0315] EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0316] SEQ ID No.4: Mouse Rα-sushi domain

[0317] GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPT

[0318] SEQ ID No.5: Human Rα-sushi domain

[0319] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP

[0320] SEQ ID No.6: Mouse Rβ extracellular domain

[0321] AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKE

[0322] SEQ ID No.7: Human Rβ extracellular domain

[0323] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLM APISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT

[0324] SEQ ID No.8: Linker segment L1

[0325] SGGGSGGGGSGGGGSGGGGSGGGSLQ

[0326] SEQ ID No.9: Linker segment L1

[0327] GGGGS

[0328] SEQ ID No.10: Linker segment L2 (MMP9)

[0329] GGGGSPVGLIGGGGGS

[0330] SEQ ID No.11: Linker segment L2 (MMP14)

[0331] GGGGSSGARYRWLTAGGGGS

[0332] SEQ ID No.12: Linker segment L2 (MMP14)

[0333] GGGGSSGRIGFLRTAGGGGS

[0334] SEQ ID No.13: Linker segment L2 (MMP14)

[0335] GGGGSSGAIGFLRTAGGGGS

[0336] SEQ ID No.14: Linker segment L2 (MMP14)

[0337] GGGGSSGRAMHMYTAGGGGS

[0338] SEQ ID No.15: Linker segment L2 (MMP14)

[0339] GGGGSSGAAMHMYTAGGGGS

[0340] SEQ ID No.16: Linker segment L2 (MMP14)

[0341] GGGGSSGRSENIRTAGGGGS

[0342] SEQ ID No.17: Linker segment L2 (MMP14)

[0343] GGGGSSGASENIRTAGGGGS

[0344] SEQ ID No.18: Linker segment L2 (MMP14)

[0345] GGGGSSGRPENIRTAGGGGS

[0346] SEQ ID No.19: Linker segment L2 (MMP14)

[0347] GGGGSSGAPENIRTAGGGGS

[0348] SEQ ID No.20: Linker segment L2 (MMP14)

[0349] GGGGSSGLISHSITAGGGGS

[0350] SEQ ID No.21: Linker segment L2 (MMP14)

[0351] GGGGSSGNLRSKLTAGGGGS

[0352] SEQ ID No.22: Linker segment L2 (MMP14)

[0353] GGGGSSGVFSIPLTAGGGGS

[0354] SEQ ID No.23: Linker segment L2 (MMP14)

[0355] GGGGSSGIKYHSLTAGGGGS

[0356] SEQ ID No.24: Mouse RA-IL15-Fc

[0357] GTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTSGGGSGGGGSGGGGSGGGGSGGGSLQNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0358] SEQ ID No.25: Human RA-IL15-Fc

[0359] ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0360] SEQ ID No.26: Mouse IL15-RA-Fc

[0361] NWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0362] SEQ ID No.27: Human IL15-RA-Fc

[0363] NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0364] SEQ ID No.28: Mouse RB-L2-IL15-RA-Fc

[0365] AVKNCSHLECFYNSRANVSCMWSHEEALNVTTCHVHAKSNLRHWNKTCELTLVRQASWACNLILGSFPESQSLTSVDLLDINVVCWEEKGWRRVKTCDFHPFDNLRLVAPHSLQVLHIDTQRCNISWKVSQVSHYIEPYLEFEARRRLLGHSWEDASVLSLKQRQQWLFLEMLIPSTSYEVQVRVKAQRNNTGTWSPWSQPLTFRTRPADPMKEGGGGSPVGLIGGGGGSNWIDVRYDLEKIESLIQSIHIDTTLYTDSDFHPSCKVTAMNCFLLELQVILHEYSNMTLNETVRNVLYLANSTLSSNKNVAESGCKECEELEEKTFTEFLQSFIRIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQGTTCPPPVSIEHADIRVKNYSVNSRERYVCNSGFKRKAGTSTLIECVINKNTNVAHWTTPSLKCIRDPSLAHYSPVPTGGGGSEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLSPGK

[0366] SEQ ID No.29: Human RB-L2-IL15-RA-Fc

[0367] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGARYRWLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0368] SEQ ID No.30: Human RB-L2-IL15-RA-Fc

[0369] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0370] SEQ ID No.31: Human RB-L2-IL15-RA-Fc

[0371] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAIGFLRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0372] SEQ ID No.32: Human RB-L2-IL15-RA-Fc

[0373] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0374] SEQ ID No.33: Human RB-L2-IL15-RA-Fc

[0375] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAAMHMYTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0376] SEQ ID No.34: Human RB-L2-IL15-RA-Fc

[0377] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRSENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0378] SEQ ID No.35: Human RB-L2-IL15-RA-Fc

[0379] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGASENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0380] SEQ ID No.36: Human RB-L2-IL15-RA-Fc

[0381] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGRPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0382] SEQ ID No.37: Human RB-L2-IL15-RA-Fc

[0383] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGAPENIRTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0384] SEQ ID No.38: Human RB-L2-IL15-RA-Fc

[0385] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGLISHSITAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0386] SEQ ID No.39: Human RB-L2-IL15-RA-Fc

[0387] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGNLRSKLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0388] SEQ ID No.40: Human RB-L2-IL15-RA-Fc

[0389] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGVFSIPLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0390] SEQ ID No.41: Human RB-L2-IL15-RA-Fc

[0391] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSSGIKYHSLTAGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0392] SEQ ID No.42: Human RB-L1-IL15-RA-Fc

[0393] AVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETL TPDTQYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDTGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMF INTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0394] The applicant's disclosure is described herein in preferred embodiments with reference to the accompanying drawings, in which like numbers represent the same or similar elements. References to "one embodiment," "an embodiment," or similar language throughout this specification mean that a particular feature, structure, or characteristic described in association with the embodiment is included in at least one embodiment of the present invention. Therefore, the appearance of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0395] The characteristics, structures or features of the disclosure of the applicant described herein can be combined in any suitable manner in one or more embodiments. A large number of specific details are described in the description herein to provide a thorough understanding of embodiments of the present invention. However, those skilled in the relevant art will recognize that the composition and / or method of the applicant can be practiced without one or more of the specific details or using other methods, components, materials, etc. In other cases, known structures, materials or operations are not shown or described in detail to avoid obscuring aspects of the present disclosure.

[0396] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure, preferred methods and materials are now described. The methods described herein may be performed in any order that is logically possible, except for the specific order disclosed.

[0397] Incorporated by Reference

[0398] References and citations are made to other documents such as patents, patent applications, patent publications, magazines, books, papers, web page contents in this disclosure. All of these documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is referred to as being incorporated herein by reference but conflicts with existing definitions, statements, or other public materials clearly set forth herein is incorporated only to the extent that no conflict is created between the incorporated material and the material of this disclosure. In the event of a conflict, the conflict will be resolved in a manner that is favorable to this disclosure as a preferred disclosure.

[0399] Equivalent

[0400] The representative examples are intended to help illustrate the present invention and are not intended and they should not be construed as limiting the scope of the present invention. In fact, for those skilled in the art, various modifications of the present invention and many other embodiments thereof will also become apparent from the full content of this document including the examples and references to the scientific and patent literature included herein, except as shown and described herein. The examples contain important additional information, examples and guidance, which can be adapted to the practice of the present invention in various embodiments of the present invention and its equivalents.

Claims

1. A fusion protein comprising: The first structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof, wherein the subunit of IL15R is an α subunit, and the amino acid sequence of the first structural unit is SEQ ID NO: 4 or SEQ ID NO: 5; The second structural unit: active interleukin 15 (IL15), wherein the IL15 is human or mouse IL15, the amino acid sequence of the mouse IL15 is SEQ ID NO: 1, and the amino acid sequence of the human IL15 is SEQ ID NO: 2; The third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein, wherein the antibody Fc fragment comprises a human Fc fragment, and the human Fc fragment is a human IgG1-Fc fragment; a linker segment L1, which covalently links the first, second and third building blocks, The fourth structural unit: the extracellular domain of the IL15 receptor β subunit, which is located at the N-terminus of the fusion protein, and the fourth structural unit is the extracellular domain of the mouse IL15 receptor β subunit or the extracellular domain of the human IL15 receptor β subunit; and a linker segment L2, which covalently connects the fourth building block and the remaining building blocks of the fusion protein, wherein the first structural unit is covalently linked to the C-terminus of the fourth structural unit, and the second structural unit is located between the first structural unit and the third structural unit, and The linker segment L2 can be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment, and the proteolytic enzyme specifically expressed in the tumor microenvironment is matrix metalloproteinase 9 or matrix metalloproteinase 14.

2. A fusion protein comprising: The first structural unit: a subunit of interleukin 15 receptor (IL15R) or a fragment thereof, wherein the subunit of IL15R is an α subunit, and the amino acid sequence of the first structural unit is SEQ ID NO: 4 or SEQ ID NO: 5; The second structural unit: active IL15, wherein the IL15 is human or mouse IL15, the amino acid sequence of the mouse IL15 is SEQ ID NO: 1, and the amino acid sequence of the human IL15 is SEQ ID NO: 2; The third structural unit: an antibody Fc fragment, which is located at the C-terminus of the fusion protein, wherein the antibody Fc fragment comprises a human Fc fragment, and the human Fc fragment is a human IgG1-Fc fragment; a linker segment L1, which covalently links the first, second and third building blocks, The fourth structural unit: the extracellular domain of the IL15 receptor β subunit, which is located at the N-terminus of the fusion protein, and the fourth structural unit is the extracellular domain of the mouse IL15 receptor β subunit or the extracellular domain of the human IL15 receptor β subunit; and a linker segment L2, which covalently connects the fourth building block and the remaining building blocks of the fusion protein, wherein the second structural unit is covalently linked to the C-terminus of the fourth structural unit, and the first structural unit is located between the second structural unit and the third structural unit, and The linker segment L2 can be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment, and the proteolytic enzyme specifically expressed in the tumor microenvironment is matrix metalloproteinase 9 or matrix metalloproteinase 14.

3. The fusion protein of any one of claims 1-2, wherein the linker segment L1 further comprises a plurality of GGGS. 4 . The fusion protein according to claim 1 , wherein the amino acid sequence of the linker segment L1 connected to the third structural unit is the amino acid sequence set forth in SEQ ID No. 9 . 5 . The fusion protein according to claim 1 , wherein the amino acid sequence of the linker segment L1 connecting the first and second structural units is the amino acid sequence set forth in SEQ ID No.

8. 6 .

6. The fusion protein according to claim 1 or 2, wherein the amino acid sequence of the IL15 receptor β subunit is the amino acid sequence set forth in SEQ ID No.

6.

7. The fusion protein according to any one of claims 1 or 2, wherein the amino acid sequence of the linker segment L2 is the amino acid sequence set forth in SEQ ID Nos. 10-23.

8. A homodimeric or heterodimeric protein comprising the fusion protein according to any one of claims 1 to 7.

9. The homodimeric or heterodimeric protein of claim 8, comprising a monomer of RA-IL15-Fc: a fusion protein of the sushi domain of the α subunit of the IL15 receptor, a linker segment L1, murine IL15, a linker segment L2, and human IgG1 Fc, and the amino acid sequence of the monomer of RA-IL15-Fc is the amino acid sequence set forth in SEQ ID No.

24.

10. The homodimeric or heterodimeric protein of claim 8, comprising a monomer of IL15-RA-Fc: a fusion protein of murine IL15, a linker segment L1, a sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1Fc, and the amino acid sequence of the monomer of IL15-RA-Fc is the amino acid sequence set forth in SEQ ID No.

26.

11. The homodimeric or heterodimeric protein of claim 8, comprising a monomer of IL15-RA-Fc: a fusion protein of human IL15, a linker segment L1, a sushi domain of the IL15 receptor α subunit, a linker segment L1, and human IgG1Fc, and the amino acid sequence of the monomer of IL15-RA-Fc is the amino acid sequence set forth in SEQ ID No.

27.

12. The homodimeric or heterodimeric protein of claim 8, comprising a monomer of RB-IL15-RA-Fc: an extracellular domain of an IL15 receptor β subunit, a linker segment L2, murine IL15, a linker segment L1, a sushi domain of an IL15 receptor α subunit, a linker segment L1, a fusion protein of human IgG1 Fc, and the amino acid sequence of the monomer of RB-IL15-RA-Fc is the amino acid sequence set forth in SEQ ID No.

28.

13. The homodimeric or heterodimeric protein of claim 8, comprising a monomer of RB-IL15-RA-Fc: an extracellular domain of an IL15 receptor β subunit, a linker segment L2, human IL15, a linker segment L1, a sushi domain of an IL15 receptor α subunit, a linker segment L1, a fusion protein of human IgG1 Fc, and the amino acid sequence of the monomer of RB-IL15-RA-Fc is any one of SEQ ID Nos. 29-41.

14. A polynucleotide encoding the fusion protein according to any one of claims 1 to 7. An expression vector comprising the polynucleotide according to claim 14 .

16. A pharmaceutical composition comprising: A fusion protein according to any one of claims 1 to 7, or a homodimer or heterodimer protein according to any one of claims 8 to 13; and Pharmaceutically acceptable excipients, carriers or diluents.

17. Use of a protein in the manufacture of a medicament for treating a tumor selected from lymphoma or colon cancer, wherein the protein comprises a therapeutically effective amount of the fusion protein of any one of claims 1 to 7, or the homodimer or heterodimer protein of any one of claims 8 to 13, or the pharmaceutical composition of claim 16.

18. The use of claim 17, further comprising one or more of chemotherapy and radiotherapy to the subject.

19. The use of claim 18, which comprises administering a chemotherapeutic agent.

20. The use of claim 18, which comprises administering radiotherapy.

21. Use of the fusion protein according to any one of claims 1 to 7, or the homodimer or heterodimer protein according to any one of claims 8 to 13 in the preparation of a drug for treating or alleviating lymphoma tumors or colon cancer tumors.

22. A cell line comprising a polynucleotide encoding the fusion protein of any one of claims 1-7.

23. A method of producing a protein, the method comprising culturing the cell line of claim 22.

24. The method of claim 23, further comprising purifying or isolating the produced protein.

25. A method for producing a protein, the method comprising: Providing an expression vector encoding the protein according to any one of claims 1 to 7; introducing the expression vector into a host cell; Cultivating the host cell in a culture medium under conditions sufficient to express the protein; and The protein is purified from the host cells or culture medium.

26. The method of claim 25, wherein the host cell is selected from the group consisting of 293F and CHO cells.

27. The method of claim 25 or 26, wherein the expression vector is introduced by transient transfection.

28. The method of claim 25 or 26, wherein the protein is purified by affinity chromatography using protein A / G or size exclusion methods.

29. An isolated protein produced by the method of any one of claims 25-28.

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