Interleukin 12 fusion proteins and compositions and therapeutic methods thereof

Abstract

To provide novel fusion proteins between interleukin 12 and a prodrug, useful in treating various diseases and disorders (e.g., hyperplasia, solid tumor or hematopoietic malignancy).SOLUTION: Provided is a fusion protein comprising: a first structural unit that is one or two subunits of IL12 selected from P35 and P40 subunits, where the first structural unit is located at the N terminus of the fusion protein; a second structural unit that is an antibody Fc fragment located at the C terminus of the fusion protein; and a first linker segment covalently linking the first structural unit and the second structural unit or covalently linking the two subunits of the first structural unit.SELECTED DRAWING: None

Description

[Technical field]

[0001] Priority claims and related applications This application claims the benefit of Chinese Application No. 201810376920.1, filed on April 25, 2018, the entire contents of which are incorporated herein by reference for all purposes.

[0002] Technical Field The present invention relates generally to novel fusion proteins and therapeutic uses. Specifically, the present invention provides novel fusion proteins and compositions of interleukin-12 and prodrugs useful in treating various diseases and disorders, such as hyperplasias, solid tumors, or hematopoietic malignancies, and methods for their preparation. [Background technology]

[0003] Interleukin 12 (IL12), also known as cytotoxic lymphocyte maturation factor (CLMF), was first identified in 1989 as a natural killer (NK) cell stimulatory factor with multiple biological activities on peripheral blood lymphocytes. L12 is produced in response to infection by various cells of the immune system, including phagocytes, B cells, and activated dendritic cells (Colombo et al. 2002 Cytokine & Growth Factor Reviews 13: 155-168). IL12 plays a key role in mediating the interaction between the innate and adaptive arms of the immune system, acting on T cells and natural killer (NK) cells, enhancing the proliferation and activity of cytotoxic lymphocytes, and promoting the production of other inflammatory cytokines, especially interferon-γ.

[0004] IL12 is a heterodimeric molecule composed of an α chain (p35 subunit) and a β chain (p40 subunit) covalently linked by disulfide bridges to form a biologically active 74 kDa heterodimer. In humans and mice, IL12 has been shown to be a potent activator of natural killer (NK) cell activity (Kobayashi, et al. 1989 J. Exp. Med. 170:827-845) and a major inducer of IFN-γ from NK and T lymphocytes, a cytokine with significant immune cell activation capabilities (Chan, et al. 1991 J. Exp. Med. 173:869-879). IFN-γ is an essential mediator of the angiogenic effects caused by IL12 (Voest, et al. 1995 J. Natl Cancer Inst. 87:581-586; Majewski, et al. 1996 J. Invest. Dermatol. 106:1114-1118).

[0005] Several studies have shown that IL12 enhances tumor cell killing mediated by immune cells specifically directed to tumor targets by antitumor antibodies (Lieberman, et al. 1991 J.Surg.Res. 50:410-415). IL12 stimulates nitric oxide production in vivo, which slows tumor progression in mice (Wigginton, et al. 1996 Cancer Res. 56:1131-1136). Endogenous IL12 production has also been reported to gradually decrease with increasing tumor burden (Handel-Fernandez, et al. 1997 J.Immunol. 158:280-286), forming a rationale for providing IL12 to cancer patients to reconstitute cell-mediated antitumor responses.

[0006] IL12 has been reported to be a potent inhibitor of tumor-induced angiogenesis (Voest, et al. 1995 supra; Majewski, et al. 1996 supra) and has demonstrated significant inhibition of tumor angiogenesis in mice in vivo mediated through IFN-γ-inducible protein-10 (IP-10; Sgadari, et al. 1996 Blood 87:3877-3882), a chemokine with potent antiangiogenic effects on the vasculature of growing tumors (Angiolillo, et al. Sci. 795:158-167; Arenberg, et al. 1996 J. Exp. Med. 184:981-992). In vitro, it inhibits the formation of tubular structures by endothelial cells (Angiolillo, et al. 1995 J. Exp. Med. 182:155-162). In vivo, induction of IP-10 by IL12 results in central tumor necrosis with surrounding vessels exhibiting intimal thickening, endothelial cell apoptosis, and partial to complete occlusion of the vessel lumen due to thrombosis (Angiolillo, et al. 1996 supra; Dias, et al. 1998 Int. J. Cancer 75:151-157). IL12 has also been shown to exert angiogenic effects through its role as a regulator of VEGF and matrix metalloproteinase (MMP) production (Dias, et al. 1998 Int. J. Cancer 78:361-365).

[0007] IL12 has shown potent antitumor effects in preclinical models; however, in clinical trials, systemic administration of recombinant IL12 caused severe side effects, including fever, gastrointestinal reactions, lymphopenia, and liver dysfunction, and patient deaths due to severe toxicity were attributed to administration of IL12 (Lasek, et al. 2014 Cancer Immunol Immunother. 63(5): 419-435).

[0008] Currently available treatments and methods for hyperplasias, solid tumors, or hematopoietic malignancies are inadequate, and there remains an urgent and continuing need for new and improved therapies to effectively treat such diseases and conditions. Summary of the Invention

[0009] 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 IL12 and their prodrugs, compositions, and methods for their preparation, which are useful for treating various diseases and disorders, such as hyperplasias, solid tumors, or hematopoietic malignancies, with reduced side effects and off-target toxicity.

[0010] In one embodiment, the present invention generally relates to a fusion protein comprising: a first structural unit: one or two subunits of IL12 selected from the P35 and P40 subunits, where the first structural unit is located at the N-terminus of the fusion protein; a second structural unit: an antibody Fc fragment, where the second structural unit is located at the C-terminus of the fusion protein; and a first linker segment covalently linking the first structural unit and the second structural unit or covalently linking the two subunits of the first structural unit.

[0011] In another aspect, the invention generally relates to homodimeric or heterodimeric proteins, including fusion proteins, as disclosed herein.

[0012] In yet another aspect, the invention generally relates to substantially purified proteins, such as the fusion proteins or fragments disclosed herein.

[0013] In yet another aspect, the invention generally relates to polynucleotides that encode a protein, such as a fusion protein or fragment thereof, disclosed herein.

[0014] In yet another aspect, the invention generally relates to expression vectors that include a polynucleotide that encodes a protein, such as a fusion protein or fragment thereof, disclosed herein.

[0015] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0016] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0017] In yet another aspect, the present invention generally relates to a method of 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 thereof, as disclosed herein, wherein the disease or condition is selected from a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0018] In yet another aspect, the invention generally relates to the use of a protein, such as a fusion protein disclosed herein or a fragment thereof, to treat or reduce a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0019] In yet another aspect, the invention generally relates to the use of polynucleotides encoding proteins, such as the fusion proteins disclosed herein, or fragments thereof, to treat or reduce a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0020] In yet another aspect, the invention generally relates to the use of a protein, such as a fusion protein or fragment thereof disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent in the manufacture of a medicament for treating or reducing a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0021] In yet another aspect, the invention generally relates to the use of a polynucleotide encoding a protein, such as a fusion protein disclosed herein, or a fragment thereof, and a pharma- ceutically acceptable excipient, carrier, or diluent, in the manufacture of a medicament for treating or reducing a disease or disorder (e.g., a hyperplasia, a solid tumor, or a hematopoietic malignancy).

[0022] In yet another aspect, the invention generally relates to cell lines comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein.

[0023] In yet another aspect, the present invention generally relates to a method for making a protein, comprising culturing a cell line, hi certain embodiments, the method further comprises purifying or isolating the produced protein, such as a fusion protein or fragment thereof disclosed herein.

[0024] In yet another aspect, the invention generally relates to a method of making a protein, the method comprising the steps of providing an expression vector encoding a protein, such as a fusion protein or fragment thereof disclosed herein, introducing the expression vector into a host cell, culturing the host cell in a medium under conditions sufficient to express the protein, and purifying the protein from the host cell or medium.

[0025] In yet another aspect, the invention generally relates to isolated proteins produced by the methods disclosed herein. [Brief description of the drawings]

[0026] [Figure 1] IL12-Fc dimeric prodrug: A schematic diagram of the structure of homodimeric IL12-Fc (Homo IL12) in tandem is shown. [Diagram 2] IL12-Fc dimer prodrug: A schematic diagram of the structure of the heterodimeric IL12-Fc in parallel form (Het IL12) is shown, where Fc-k is the short form for Fc-knob and Fc-h is the short form for Fc-hole. [Diagram 3] FIG. 1 shows a schematic diagram of the structure of homodimeric IL12-Rβ1 dimer prodrug (Homo-R1). [Figure 4] FIG. 1 shows a schematic diagram of the structure of homodimeric IL12-Rβ2 dimer prodrug (Homo-R2). [Diagram 5] Schematic diagram of the structure of heterodimeric IL12-Rβ1 / Rβ2 dimer prodrug (Heto-R1 / R2), where Fc-k is the short form of Fc-knob and Fc-h is the short form of Fc-hole. [Figure 6] Schematic diagram of the structure of heterodimeric IL12-Rβ1 dimer prodrug (Hetero-R1), where Fc-k is short for Fc-knob and Fc-h is short for Fc-hole. [Figure 7] Schematic diagram of the structure of heterodimeric IL12-Rβ2 dimer prodrug (Hetero-R2), where Fc-k is short for Fc-knob and Fc-h is short for Fc-hole. [Figure 8A] 1 shows exemplary data of SDS-PAGE electrophoresis results of the expression of the seven fusion proteins of FIGS. 1-7. [Figure 8B] 1 shows exemplary data of SDS-PAGE electrophoresis results of the expression of the seven fusion proteins of FIGS. 1-7. [Figure 9]Exemplary data show that injection of IL12-Fc that is not bound to the IL12 receptor completely eliminates MC38 tumors, with Het IL12 having a stronger elimination effect than Homo IL12. [Figure 10-1] Exemplary data are presented showing that Het IL12 has higher cytotoxicity than Homo IL12. [Figure 10-2] Exemplary data are presented showing that Het IL12 has higher cytotoxicity than Homo IL12. [Figure 11] Exemplary data are presented showing that Het R1, Het R2, and Het-R1 / R2 all effectively eliminate MC38 tumors. [Figure 12A] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12B] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12C] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12D] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12E] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12F] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 12G] Exemplary data are presented showing that a Het IL12 prodrug conjugated to the IL12 receptor has fewer side effects when administered systemically. [Figure 13] Exemplary data of SDS-PAGE electrophoresis results of human Het-R1 and Het-R1 / R2 expression with and without digestion with MMP14 are shown. [Figure 14] Exemplary data are shown showing that (A) human Het-R1 after MMP14 digestion exhibits similar activity to Het IL12 and recombinant IL12 in vitro, and (B) human Het-R1 / R2 after zMMP14 digestion exhibits similar activity to Het IL12 and recombinant IL12 using the HEK Blue-IL12 reporter cell line.

[0027] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following terms are intended to have the following meanings, unless otherwise defined, according to the context in which the term is found:

[0028] When trade names are used herein, they include formulations of the trade name product, generic drugs, and the active pharmaceutical ingredients of the trade name product, unless otherwise specified.

[0029] Ranges provided herein are understood to be shorthand expressions for values ​​within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0030] As used herein, "at least" a particular value is understood to mean that value and all values ​​greater than that value.

[0031] As used herein, "two or more" 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 in between.

[0032] In the specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0033] As used herein, unless specifically stated otherwise or clear from the context, the term "about" is understood to be within the normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" may 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. Unless otherwise clear from the context, all numerical values ​​provided herein may be modified by the term "about".

[0034] As used herein, unless specifically stated otherwise or clear from the context, the term "or" is to be understood as being inclusive.

[0035] When used to define compositions and methods, the term "comprising" shall mean that the compositions and methods include the recited elements but do not exclude other elements. When used to define compositions and methods, the term "consisting essentially of" shall mean that the compositions and methods include the recited elements and exclude other elements of essential importance to the compositions and methods. For example, the term "consisting essentially of" refers to administration of pharmacologically active agents that are expressly recited and excludes pharmacologically active agents that are not expressly recited. The term "consisting essentially of" does not exclude pharmacologic inactive or inactive agents, such as pharmacologic acceptable excipients, carriers, or diluents. The term "consisting of" when used to define compositions and methods shall mean excluding trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0036] As used herein, the term "agonist" refers to a compound that can combine with a receptor to produce a cellular response. An agonist may be a ligand that directly binds to a receptor. Alternatively, an agonist may bind to a receptor indirectly, for example, by (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise effecting the modification of another compound so that the other compound directly binds to the receptor.

[0037] As used herein, the term "antagonist" refers to a compound that competes with an agonist or inverse agonist for binding to a receptor, thereby blocking the action of the agonist or inverse agonist on the receptor. However, antagonists do not affect constitutive receptor activity.

[0038] As used herein, the term "antibody" refers to a molecule capable of binding to an epitope or antigenic determinant. The term is intended to include whole antibodies and antigen-binding fragments thereof. The term encompasses polyclonal, monoclonal, chimeric, Fab, Fvs, single chain antibodies and single or multiple immunoglobulin variable chains, or CDR domain designs, as well as bispecific and multispecific antibodies. Antibodies may be from any animal. Preferably, the antibodies are mammalian, e.g., human, murine, rabbit, goat, guinea pig, camel, horse, etc., or other suitable animals. Antibodies may recognize polypeptide or polynucleotide antigens. The term includes, for example, antigen-binding fragments of immunoglobulins, active fragments including the variable and / or constant regions of the heavy chain, the variable and / or constant regions of the light chain, the complementarity determining regions (cdrs), and the framework regions. The term includes polyclonal and monoclonal antibody preparations, as well as hybrid antibodies, modified antibodies, chimeric antibodies, hybrid antibody molecules, preparations including F(ab)2 and F(ab) fragments; Fv molecules (e.g., non-covalent heterodimers), dimeric and trimeric antibody fragment constructs; minibodies, humanized antibody molecules, and functional fragments derived from such molecules, where such fragments retain specific binding.

[0039] As used herein, the term "antigen" refers to any substance that causes the immune system to generate antibodies or a specific cell-mediated immune response against it. Disease-associated antigen refers to any substance associated with any disease that causes the immune system to generate antibodies or a specific cell-mediated immune response against it. An antigen can be recognized by the immune system and / or induce a humoral and / or cellular immune response that leads to the activation of B- and / or T-lymphocytes. An antigen can have one or more epitopes (B- and / or T-cell epitopes). An antigen preferably reacts with its corresponding antibody or TCR, typically in a highly selective manner, and not with the multitude of other antibodies or TCRs that may be elicited by other antigens. An antigen as used herein may also be a mixture of multiple individual antigens.

[0040] As used herein, the term "biologically active" or "biologically active" refers to those that have structural, regulatory, or biochemical functions of naturally occurring molecules, or any functions related or associated with metabolic or physiological processes. Biologically active polypeptides or fragments thereof include those that can participate in a biological process or reaction and / or produce a desired effect. Biological activity can include improved desired activity or decreased undesirable activity. For example, an entity exerts biological activity if it participates in molecular interactions with other molecules, has therapeutic value in alleviating a disease state, has preventive value in inducing an immune response, or has diagnostic and / or prognostic value in determining the presence of a molecule. Biologically active proteins or polypeptides can be naturally occurring or can be synthesized, for example, by recombinant or chemical synthesis, from known components, and can include heterologous components.

[0041] As used herein, the terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, sarcoma, blastoma, and leukemia. Specific examples of such cancers include squamous cell carcinoma, lung cancer, pancreatic cancer, cervical cancer, bladder cancer, liver cancer, breast cancer, colon cancer, and head and neck cancer.

[0042] As used herein, the term "cell" refers to any prokaryotic, eukaryotic, primary or immortalized cell line, any group of such cells, such as a tissue or organ. Preferably, the cells are of mammalian (e.g., human) origin and can be infected with one or more pathogens.

[0043] As used herein, the term "co-administration" refers to the presence of two pharmacological agents in the blood at the same time. The two pharmacological agents can be administered simultaneously or sequentially.

[0044] As used herein, the term "co-expression" is intended to mean that two different polypeptides are expressed simultaneously in a host cell such that the two polypeptides can interact or bind to form a complex either in the host cell or in the host cell culture medium.

[0045] As used herein, the term "disease" or "disorder" refers to a pathological condition, e.g., one that can be identified as deviating from a healthy or normal state by symptoms or other distinguishing factors. The term "disease" includes disorders, syndromes, conditions, and injuries. Diseases include, but are not limited to, proliferative diseases, inflammatory diseases, immune diseases, metabolic diseases, infectious diseases, and ischemic diseases.

[0046] As used herein, the term "effective amount" of active agent refers to the amount sufficient to elicit desired biological response.As understood by those skilled in the art, the effective amount of the compound of the present invention may vary depending on such factors as desired biological endpoint, pharmacokinetics of the compound, disease being treated, administration form and patient.

[0047] As used herein, the term "expression of a nucleic acid molecule" refers to the conversion of the information contained in a nucleic acid molecule into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a peptide or polypeptide produced by translation of an mRNA. Gene products further include RNA modified by processes such as capping, polyadenylation, methylation, and editing; and proteins modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

[0048] As used herein, the term "host cell" refers to an individual cell or cell culture that can be or has been the recipient of any recombinant vector or isolated polynucleotide. A host cell can be a transfected, transformed, transduced, or infected cell of any origin, including prokaryotic, eukaryotic, mammalian, avian, insect, plant, or bacterial cells, or any origin that can be used to propagate a nucleic acid described herein. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutations and / or changes. A host cell includes cells that have been transfected or infected in vitro or in vitro with a recombinant vector or polynucleotide of the invention. A host cell containing a recombinant vector of the invention may also be referred to as a "recombinant host cell."

[0049] Host cells include, but are not limited to, mammalian, plant, insect, fungal, and bacterial cells. Bacterial cells preferably include, but are not limited to, Gram-positive cells, such as those of the genus Bacillus, Streptomyces, and Staphylococcus, and Gram-negative cells, such as those of the genus Escherichia and Pseudomonas. Fungal cells preferably include yeast cells, such as those of Saccharomyces, Pichia pastoris, and Hansenula polymorpha. Insect cells include, but are not limited to, Drosophila cells, and Sf9 cells. Plant cells include, inter alia, cells from crop plants, such as cereals, medicinal or ornamental plants, or bulbs. Mammalian cells suitable for the present invention include epithelial cell lines (such as porcine), osteosarcoma cell lines (such as human), neuroblastoma cell lines (such as human), epithelial carcinoma cell lines (such as human), glial cell lines (such as mouse), liver cell lines (such as monkey), CHO cells (Chinese Hamster Ovary), COS cells, BHK cells, cells HeLa, 911, AT1080, A549, 293 or PER.C6, human ECCs NTERA-2 cells, mESCs lines D3 cells, human embryonic stem cells such as HS293 and BGV01, SHEF1, SHEF2 and HS181, cells NIH3T3, 293T, REH, and MCF-7 as well as hMSCs cells.

[0050] As used herein, the term "Fc" refers to a molecule or sequence consisting of a sequence of non-antigen-binding fragments of a whole antibody, whether in monomeric or multimeric form. The original immunoglobulin source of native Fc is preferably of human origin and can be any immunoglobulin (e.g., IgG1, IgG2). Native Fc is composed of monomeric polypeptides that can be linked into dimeric or multimeric forms by covalent (i.e., disulfide bonds) and non-covalent bonds. The number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4, depending on the class (e.g., IgG, IgA, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2).

[0051] As used herein, the term "Fc domain" or "Fc region" is meant to refer to an immunoglobulin heavy chain "fragment crystallizable" region. Generally, an Fc domain can interact with a second Fc domain to form a dimeric complex. An Fc domain can bind cell surface receptors called Fc receptors and / or proteins of the complement system, or may be modified to reduce or enhance these binding activities. Fc domains may be derived from IgG, IgA, IgD, IgM, or IgE antibody isotypes and are involved in immune activities including oxonization, cell lysis, degranulation of mast cells, basophils, eosinophils, and other Fc receptor-dependent processes; activation of the complement pathway; and affecting protein stability in vivo.

[0052] "Fc domain" as defined herein encompasses native Fc and Fc variant molecules and sequences. As with Fc variants and native Fcs, the term "Fc domain" encompasses the molecule in monomeric or multimeric form, whether extracted from a whole antibody or produced by recombinant gene expression or other means.

[0053] Fc fusion proteins have been reported that combine the Fc region of IgG with domains of other proteins, such as various cytokines and soluble receptors (e.g., Capon et al. 1989 Nature 337:525-531; Chamow et al. 1996 Trends Biotechnol. 14:52-60; US Pat. Nos. 5,116,964 and 5,541,087).

[0054] The use of Fc fusions is known in the art (e.g., U.S. Patent Nos. 7,754,855; 5,480,981; 5,808,029; WO7 / 23614; WO98 / 28427, and references cited herein). Fc fusion proteins can include mutant Fc molecules (e.g., as described in U.S. Patent No. 7,732,570). Fc fusion proteins can be soluble in plasma or can bind to the cell surface of cells that have specific Fc receptors.

[0055] As used herein, the term "Fc variant" refers to a molecule or sequence that has been modified from a native Fc but contains the binding site of the salvage receptor FcRn. International Applications WO 97 / 34631 (published September 25, 1997) and WO 96 / 32478 describe exemplary Fc variants and their interaction with the salvage receptor and are incorporated herein by reference. Thus, the term "Fc variant" includes a molecule or sequence that has been humanized from a non-human native Fc. Additionally, the native Fc contains sites that can be removed to provide structural features or biological activity that are not required for the fusion molecules of the present invention. Thus, in certain embodiments, the term "Fc variant" includes molecules or sequences that lack one or more native Fc sites or residues that affect or are involved in (1) disulfide bond formation, (2) incompatibility with a selected host cell, (3) N-terminal heterogeneity upon expression in a selected host cell, (4) glycosylation, (5) interaction with complement, (6) binding to Fc receptors other than the salvage receptor, or (7) antibody-dependent cellular cytotoxicity (ADCC). Fc variants are described in further detail below.

[0056] As used herein, the term "fusion protein" refers to a polypeptide comprising two or more regions from different or heterologous proteins covalently linked (i.e., "fused") by recombinant, chemical or other suitable methods. If desired, the fusion molecule can be fused at one or more sites via a peptide or other linker segment or sequence. For example, one or more peptide linkers can be used to aid in the construction of the fusion protein.

[0057] As used herein, the term "GC content" refers to the percentage of a nucleic acid sequence that is composed of deoxyguanosine (G) and / or deoxycytidine (C) deoxyribonucleoside, or guanosine (G) and / or cytidine (C) ribonucleoside residues.

[0058] As used herein, the term "high dose" means at least 5% (e.g., at least 10%, 20%, 50%, 100%, 200%, or even 300%) more than the highest standard recommended dose of a particular compound for the treatment of any human disease or condition.

[0059] As used herein, the term "immune response" refers to the process by which immune cells are stimulated and / or recruited from blood to lymphoid and non-lymphoid tissues through a multifactorial process that includes distinct adhesion and / or activation steps. Activating conditions cause the release of cytokines, growth factors, chemokines, and other factors, upregulate the expression of adhesion and other activation molecules on immune cells, promote chemotaxis and simultaneous adhesion, morphological changes, and / or extravasation through tissues, increase cell proliferation and cytotoxic activity, stimulate antigen presentation, and cause other phenotypic changes including the generation of memory cell types. Immune response is also meant to refer to the activity of immune cells to suppress or modulate the inflammatory or cytotoxic activity of other immune cells. Immune response refers to the activity of immune cells in vivo or in vitro.

[0060] The term "identity" or percent "identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences having a predetermined percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identical over a designated region (e.g., of an IL12 sequence or an IL12R sequence), preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters, described below, or by manual alignment and visual inspection. Such sequences are said to be "substantially identical." This definition may also refer to or apply to the complement of a test sequence. This definition further includes sequences that have deletions and / or additions, as well as sequences that have substitutions. As described below, preferred algorithms can account for gaps, etc. Preferably, identity exists over sites that are at least about 25, 50, 75, 100, 150, 200 amino acids or nucleotides in length, and often over sites that are 225, 250, 300, 350, 400, 450, 500 amino acids or nucleotides in length, or over the full-length amino acid or nucleic acid sequence.

[0061] For sequence comparison, typically, one sequence acts as a reference sequence, and test sequence is compared to it.Using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequent coordinates are designated as necessary, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence to reference sequence based on program parameters.

[0062] A preferred example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. 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 on the World Wide Web at ncbi.nlm.nih.gov / . Both default parameters or other non-default parameters can be used. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989)) of alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0063] As used herein, the term "inhibit" refers to any measurable decrease in biological activity. Thus, as used herein, "inhibit" or "inhibition" may refer to a percentage of normal activity levels.

[0064] As used herein, the term "Interleukin-12" or "IL12" refers to a polypeptide having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to a biologically active mammalian native IL12 amino acid sequence, meaning that a mutated protein ("mutein") has similar functionality (75% or greater) as the native IL12 protein in at least one functional assay.

[0065] Exemplary functional assays of IL12 polypeptide include, for example, inducing the production of interferon-γ (IFN-γ) by T cells or natural killer (NK) cells, and promoting the differentiation of T helper-1 (Th1) cells. Helper T cells differentiated into Th1 cells can be identified by secretion of IFN-γ. For example, IFN-γ secreted by T cells or NK cells stimulated with IL-12 can be conveniently detected, for example, in serum or cell culture supernatant using ELISA. ELISA methods and techniques are well known in the art, and kits for detecting IFN-γ are commercially available (e.g., R&D Systems, Minneapolis, Minn.; Peprotech, Rocky Hill, NJ; and Biosource Intl., Camarillo, Calif.). See also, Coligan, et al., Current Methods in Immunology, 1991-2006, John Wiley & Sons; Harlow and Lane, Using Antibodies: A Laboratory Manual, 1998, Cold Spring Harbor Laboratory Press; and The ELISA Guidebook, Crowther, ed., 2000, Humana Press.

[0066] As used herein, the term "isolated" molecule (such as a polypeptide or polynucleotide) is one that has been engineered to exist in greater concentrations than in nature or has been removed from its original environment. For example, a subject antibody is isolated, purified, substantially isolated, or substantially purified when at least 10%, or 20%, or 40%, or 50%, or 70%, or 90% of the non-subject antibody material with which it is naturally associated has been removed. For example, a polynucleotide or polypeptide that is 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." Additionally, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA molecules and RNA molecules. Isolated nucleic acid molecules further include molecules that are synthetically produced. In addition, vector molecules contained in a recombinant host cell are also isolated. Thus, not all "isolated" molecules need to be "purified."

[0067] As used herein, "linker" or "linking segment" refers to a molecule or group that connects two other molecules or groups. A peptide linker may allow the connected molecules or groups to acquire 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 approximately 100 amino acids.

[0068] The components of the fusion protein, such as the cytokine or other bioactive molecule and any peptide linker, can be organized in almost any way, provided that the fusion protein has the intended function. In particular, each component of the fusion protein can be spaced apart from another component by at least one suitable peptide linker segment or sequence, if desired. In addition, the fusion protein can include, for example, a tag to facilitate the modification, identification, and / or purification of the fusion protein. Specific fusion proteins are given in the examples below.

[0069] As used herein, the term "low dose" means at least 5% (e.g., at least 10%, 20%, 50%, 80%, 90%, or even 95%) less 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 drug formulated for administration by inhalation will be different from a low dose of the same drug formulated for oral administration. For example, a low dose of a drug formulated for administration by inhalation will be different from a low dose of the same drug formulated for oral administration.

[0070] As used herein, the term "medium" or "media" includes any culture medium, solution, solid, semi-solid, or rigid support that can support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli host cells, or Pseudomonas host cells, and cell contents. Thus, the term can encompass the medium in which the host cell was grown, e.g., the medium into which the polypeptide was secreted, including the medium either before or after the growth step. The term may further encompass buffers or reagents that include host cell lysates, such as when the polypeptide is produced intracellularly and the host cells are lysed or destroyed to release the polypeptide.

[0071] As used herein, the term "modulate" refers to directly or indirectly increasing or decreasing, stimulating, inhibiting, interfering, or blocking 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, stimulates, inhibits, interferes, or blocks, the measured activity of a polypeptide or polynucleotide when compared to a suitable control. For example, a "modulator" can bind to a target with measurable affinity and / or activate or inhibit the target, or directly or indirectly affect the normal regulation of receptor activity.

[0072] The term "operably linked" refers to a functional linkage between a first and a second nucleic acid sequence such that the first and second nucleic acid sequences are transcribed into a single nucleic acid sequence. Operatively linked nucleic acid sequences need not be physically adjacent to each other. The term "operably linked" further refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or an array of transcription factor binding sites) and a transcribable nucleic acid sequence, where the expression control sequence directs the transcription of the nucleic acid corresponding to the transcribable sequence.

[0073] As used herein, the term "pharmaceutically acceptable" excipient, carrier, or diluent refers to a pharma- ceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating substance, involved in carrying or transporting the subject pharmaceutical agent from one organ or body part to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of substances which can function as pharma- ceutically 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; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffers; and other non-toxic compatible substances utilized in pharmaceutical preparations. 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 composition.

[0074] As used herein, the terms "polynucleotide," "nucleic acid molecule," "nucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably herein to refer to polymeric forms of nucleotides, including ribonucleotides and deoxyribonucleotides of any length. They can include both double-stranded, single-stranded, or triple-helical sequences, including, but not limited to, cDNA:mRNA from viral, prokaryotic, and eukaryotic sources; 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 term further captures sequences that contain any of the known base analogs of DNA and RNA. Nucleotides may also be referred to by their commonly accepted single-letter codes.

[0075] Polynucleotides are not limited to polynucleotides occurring in nature, but also include polynucleotides in which unnatural nucleotide analogs and internucleotide linkages occur. Nucleic acid molecules can include modified nucleic acid molecules (e.g., modified bases, sugars, and / or internucleotide linkers). Non-limiting examples of this type of unnatural structure include polynucleotides in which the sugar is different from ribose, polynucleotides in which phosphodiester linkages 3'-5' and 2'-5' occur, polynucleotides in which inverted linkages (3'-3' and 5'-5') occur, and branched structures. Similarly, polynucleotides of the invention include unnatural internucleotide linkages such as peptide nucleic acids (PNAs), locked nucleic acids (LNAs), C1-C4 alkyl phosphonate linkages of methyl phosphonate, phosphoramidates, C1-C6 alkyl phosphotriesters, phosphorothioates, and phosphorodithioates types. In any case, polynucleotides of the invention maintain the ability to hybridize to target nucleic acids in a manner similar to naturally occurring polynucleotides.

[0076] Unless otherwise specified or apparent from the context, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions) and the complementary sequence and the sequence explicitly stated. 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-base and / or deoxyinosine residues. (Batzer et al. 1991 Nucleic Acid Res. 19:5081; Ohtsuka et al. 1985 J. Biol. Chem. 260:2605-2608; Rossolini et al. 1994 Mol. Cell. Probes 8:91-98.)

[0077] As used herein, the terms "prevent", "preventing" or "prevention" refer to a method for preventing, delaying, avoiding or halting the onset, incidence, severity or recurrence of a disease or condition. For example, a method is considered to be preventative if there is a reduction or delay in the onset, incidence, severity or recurrence of the 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. A disclosed method is considered to be preventative if there is a reduction or delay in the onset, incidence, severity or recurrence of one or more symptoms of the disease or condition in a subject susceptible to the disease or condition after receiving the method, compared to the progression of the subject before receiving treatment. The reduction or delay in the onset, incidence, severity or recurrence of osteoporosis can be about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount therebetween.

[0078] Prevention and the like does not mean to prevent a subject from contracting a particular disease or disorder. Prevention may require multiple administrations. Prevention can include prevention of recurrence of disease in a subject in which all disease symptoms have been eliminated, or prevention of recurrence in a recurrent disease.

[0079] As used herein, the term "promoter" refers to a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of an operably linked downstream (3' direction) coding sequence. A promoter sequence includes the minimum number of bases or elements required to initiate transcription of a gene of interest at a level that is more detectable than conventional. Included within the promoter sequence may be a transcription initiation site and a protein binding domain (consensus sequence) involved in the binding of RNA polymerase. Eukaryotic promoters often, but not always, include "TATA" boxes and "CAT" boxes. Promoters include those that are naturally contiguous with a nucleic acid molecule and those that are not naturally contiguous with a nucleic acid molecule. In addition, the term "promoter" includes inducible promoters, conditionally active promoters such as the cre-lox promoter, constitutive promoters, and tissue-specific promoters.

[0080] As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included in the definition. Both full-length proteins and fragments thereof are encompassed in the definition. The term includes post-expression modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like. Additionally, a polypeptide may refer to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the protein maintains the desired activity. Such modifications may be intentional or accidental. Amino acids may be referred to herein by either their commonly known three letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0081] As used herein, the term "purified" refers to a protein that 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., a native cell, or in the case of recombinantly produced proteins, a host cell. Proteins that may be substantially free of cellular material include preparations of the protein that have less than about 30%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating proteins. When a protein or variant thereof is recombinantly produced by a host cell, the protein may be present at about 30%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or less than about 1% of the dry weight of the cell. When the protein or variant thereof is recombinantly produced by a host cell, the protein may be present in the culture medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about 750 mg / L, about 500 mg / L, about 250 mg / L, about 100 mg / L, about 50 mg / L, about 10 mg / L, or about 1 mg / L or less of dry weight of the cells. Thus, a "substantially purified" protein may have a purity level of at least about 80%, specifically at least about 85%, more specifically at least about 90%, at least about 95%, at least about 99% or more, as determined by suitable methods such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0082] After their preparation, the proteins and prodrugs of the invention are preferably isolated and / or purified to obtain compositions that contain 80% or more by weight ("substantially pure"), and then used or formulated as described herein. In certain embodiments, the compounds of the invention are greater than 95% pure.

[0083] As used herein, the term "receptor" refers to a protein, including a glycoprotein or a fragment thereof, that can interact with another molecule called a ligand. A ligand may belong to any class of biochemical or chemical compound. A ligand is typically an extracellular molecule that, upon binding to the receptor, typically initiates a cellular response, such as the initiation of a signal transduction pathway. A receptor is not necessarily a membrane-bound protein.

[0084] As used herein, the term "recombinant" with respect to a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, meaning that the polynucleotide is not related by its origin or manipulation to all or a portion of the polynucleotide 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" with respect to a host cell means a host cell into which a recombinant polynucleotide has been introduced.

[0085] As used herein, the term "recombinant virus" refers to a virus that has been genetically modified by the hand of man. This phrase covers any virus known in the art.

[0086] As used herein, the term "sample" refers to a sample from a human, animal, or research sample, such as a cell, tissue, organ, fluid, gas, aerosol, slurry, colloid, or coagulate. A "sample" may be tested in vivo, for example, without removal from a human or animal, or in vitro. A "sample" may be tested after processing, for example, by histological methods. A "sample" may also refer to cells, including, for example, a fluid or tissue sample, or cells separated from a fluid or tissue sample. A "sample" may also refer to cells, tissues, organs, or fluids taken fresh from a human or animal, or cells, tissues, organs, or fluids that have been processed or preserved.

[0087] As used herein, the term "soluble" refers to fusion molecules, particularly fusion proteins, that do not readily sediment in aqueous buffers, e.g., low G-force centrifugation (e.g., less than about 30,000 revolutions per minute in a standard centrifuge) from cell culture media. A fusion molecule is soluble if it remains in aqueous solution at or near neutral pH, at temperatures greater than about 5-37°C, in the presence of low or no concentrations of anionic or nonionic detergents. Under such conditions, soluble proteins often have low sedimentation values, e.g., less than about 10-about 50 Svedberg units.

[0088] The aqueous solutions referred to herein are typically within a pH range of about 5-9, and typically have a buffer compound to establish a pH in the ionic strength range of about 2 mM-500 mM. Optionally, protease inhibitors or mild non-ionic detergents are added. Additionally, carrier proteins (e.g., bovine serum albumin) may be added as needed. Exemplary aqueous buffers include standard phosphate buffered saline, Tris buffered saline, or other well-known buffers and cell culture media formulations.

[0089] As used herein, the term "stimulate" or "stimulating" refers to increasing, amplifying, enhancing, boosting a physiological activity, such as an immune response. Stimulation can be a positive change in quality. For example, there can be an increase of 5%, 10%, 25%, 50%, 75%, or even 90-100%. Other exemplary increases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0090] As used herein, the terms "subject" and "patient" are used interchangeably to refer to a living animal, human or non-human. The subject may be a mammal. The terms "mammal" or "mammalian" refer to any animal within the taxonomic classification Mammalia. A mammal may be a human or a non-human mammal, such as a dog, cat, pig, cow, sheep, goat, horse, rat, and mouse. The term "subject" does not exclude individuals who are completely normal or normal in all respects with respect to a disease or condition.

[0091] As used herein, the term "suppress" or "suppressing" refers to lowering, attenuating, decreasing, stopping, or stabilizing a biological activity, such as an immune response. The suppression may be a negative property change. For example, there may be a 5%, 10%, 25%, 50%, 75%, or even 90-100% decrease. Exemplary decreases include 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, or even 100-fold.

[0092] As used herein, the term "therapeutically effective amount" refers to a dosage of a therapeutic agent sufficient to achieve the intended therapeutic effect with minimal or no undesirable side effects. A therapeutically effective amount can be readily determined by one of ordinary skill in the art, for example, by initially administering a low dose of a pharmacological agent and then gradually increasing the dosage until the desired therapeutic effect is achieved with minimal or no undesirable side effects.

[0093] As used herein, the term "transfected" means having introduced DNA or RNA, with or without the use of an associated facilitating agent such as lipofectamine. Methods of transfection known in the art include, for example, calcium phosphate transfection, DEAE-dextran transfection, protoplast fusion, electroporation, and lipofection.

[0094] As used herein, the term "treat" or "treating" a disease or disorder refers to a method of reducing, delaying, or ameliorating such a condition, or one or more symptoms of such a disease or condition, before or after its onset. Treatment may be directed to one or more effects or symptoms of the disease and / or underlying pathology. Said treatment may be any alleviation, including but not limited to, complete disappearance of the disease or its symptoms. The degree of such alleviation or prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique, compared to an equivalent untreated control.

[0095] As used herein, the term "tumor" refers to any malignant or neoplastic cell.

[0096] As used herein, the term "vector" refers to a nucleic acid molecule capable of transferring genetic material to a host cell or organism. A vector may be composed of either DNA or RNA. A vector carries its own origin of replication, one or more unique recognition sites for restriction endonucleases that can be used to insert foreign DNA, usually a selectable marker such as a gene encoding antibiotic resistance, and often a recognition sequence (e.g., a promoter) for the expression of the inserted DNA. Common vectors include plasmid vectors and phage vectors.

[0097] Any composition or method disclosed herein can be combined with any one or more of the other compositions and methods provided herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0098] The present invention provides a novel fusion protein and its therapeutic use. More specifically, the present invention provides a novel fusion protein of IL12 and its prodrug, composition and preparation method thereof. These are useful for treating various diseases and disorders, such as hyperplasia, solid tumor, or hematopoietic malignancy, with reduced off-target toxicity and side effects during treatment.

[0099] In one aspect, the present invention generally relates to a fusion protein comprising a first structural unit which is one or more subunits of IL12 selected from the P35 and P40 subunits, the first structural unit being located at the N-terminus of the fusion protein, a second structural unit which is an antibody Fc fragment, the second structural unit being located at the C-terminus of the fusion protein, and a first linker segment which covalently links the first structural unit to the second structural unit or which covalently links two subunits of the first structural unit.

[0100] In certain embodiments of the fusion protein, the P35 and P40 subunits are derived from a mammal selected from the group consisting of human, monkey, mouse, dog, rat, cow, pig, and sheep.

[0101] In some embodiments of the fusion protein, the P35 and P40 subunits are from human. In some embodiments of the fusion protein, the P35 and P40 subunits are from monkey. In some embodiments of the fusion protein, the P35 and P40 subunits are from mouse. In some embodiments of the fusion protein, the P35 and P40 subunits are from dog. In some embodiments of the fusion protein, the P35 and P40 subunits are from a mammal selected from rat. In some embodiments of the fusion protein, the P35 and P40 subunits are from bovine. In some embodiments of the fusion protein, the P35 and P40 subunits are from porcine. In some embodiments of the fusion protein, the P35 and P40 subunits are from ovine.

[0102] Any suitable antibody Fc fragment may be utilized.

[0103] In some embodiments of the fusion protein, the antibody Fc fragment comprises a human Fc fragment. In some embodiments, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No.5.

[0104] In one embodiment, the antibody Fc fragment comprises a human IgG1. In one embodiment, the antibody Fc fragment comprises the amino acid sequence set forth in SEQ ID No.6.

[0105] In certain embodiments, the human IgG1 is a human Fc-knob or a human Fc-hole.

[0106] In one embodiment, the human IgG1 comprises the amino acid sequence set forth in SEQ ID No.7.

[0107] In one embodiment of the fusion protein, the mouse P35 subunit has the amino acid sequence set forth in SEQ ID No.3.

[0108] In one embodiment of the fusion protein, the human P35 subunit has the amino acid sequence set forth in SEQ ID No.4.

[0109] In one embodiment of the fusion protein, the mouse P40 subunit has the amino acid sequence set forth in SEQ ID No.1.

[0110] In one embodiment of the fusion protein, the human P40 subunit has the amino acid sequence set forth in SEQ ID No.2.

[0111] In one embodiment of the fusion protein, the first linker segment L1 has the amino acid sequence set forth in SEQ ID No.12.

[0112] In certain embodiments, the fusion protein further comprises a signal peptide, eg, modified at the N-terminus of the first structural unit.

[0113] In one embodiment, the N-terminally modified signal peptide SP1 of the mouse P35 subunit comprises the amino acid sequence set forth in SEQ ID No.27.

[0114] In one embodiment, the N-terminally modified signal peptide SP1 of the mouse P35 subunit comprises the amino acid sequence set forth in SEQ ID No.28.

[0115] In one embodiment, the N-terminally modified signal peptide SP2 of the mouse P40 subunit comprises the amino acid sequence set forth in SEQ ID No.29.

[0116] In one embodiment, the N-terminally modified signal peptide SP2 of the human P40 subunit comprises the amino acid sequence set forth in SEQ ID No.30.

[0117] In some embodiments, the fusion protein further comprises a portion of an interleukin 12 receptor (IL12R) covalently linked to the N-terminus of the first structural unit by a second linker segment (L2). In some embodiments, the IL12R is selected from Rβ1 and Rβ2.

[0118] In one embodiment, mouse Rβ1 comprises the amino acid sequence set forth in SEQ ID No.8, and mouse Rβ2 comprises the amino acid sequence set forth in SEQ ID No.10.

[0119] In one embodiment, human Rβ1 comprises the amino acid sequence set forth in SEQ ID No.9, and human Rβ2 comprises the amino acid sequence set forth in SEQ ID No.11.

[0120] In certain embodiments of the fusion protein, the second linker segment, L2, may be recognized and hydrolyzed by a proteolytic enzyme specifically expressed in the tumor microenvironment.

[0121] In certain embodiments, the proteolytic enzyme specifically expressed in the tumor microenvironment is a matrix metalloprotease, such as matrix metalloprotease 14 (MMP14).

[0122] In one embodiment of the fusion protein, the second linker segment L2 comprises an amino acid sequence set forth in SEQ ID Nos. 13-26.

[0123] In one embodiment of the fusion protein, the C-terminus of IL12R is linked to the N-terminus of the first structural unit via L2, and the C-terminus of the first structural unit and the N-terminus of the second structural unit are linked by L1. When the first structural unit comprises two subunits, the C-terminus of the first subunit and the N-terminus of the second subunit are linked by a linker segment L1.

[0124] In another aspect, the invention generally relates to homodimeric or heterodimeric proteins, including fusion proteins, as disclosed herein.

[0125] In one embodiment, the homodimeric or heterodimeric protein is a monomeric homodimer having an amino acid sequence as set forth in, for example, SEQ ID No. 31, including a fusion protein of mouse P40 subunit, L1 linker, mouse P35 subunit, L1 linker, and human IgG1.

[0126] In one embodiment, the homodimeric or heterodimeric protein is a monomeric homodimer having an amino acid sequence as set forth in, for example, SEQ ID No. 32, including a fusion protein of a human P40 subunit, an L1 linker, a human P35 subunit, an L1 linker, and a human IgG1.

[0127] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer: a fusion protein comprising a mouse P40 subunit, an L1 linker, and a human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 33, and a second monomer: a fusion protein comprising a mouse P35 subunit, an L1 linker, and a human Fc-hole, and having, for example, the amino acid sequence structure set forth in SEQ ID No. 35.

[0128] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer: a fusion protein comprising a human P40 subunit, an L1 linker, and a human Fc-knob, and having the amino acid sequence set forth in SEQ ID No. 34, and a second monomer: a fusion protein comprising a human P35 subunit, an L1 linker, and a human Fc-hole, and having, for example, the amino acid sequence structure set forth in SEQ ID No. 36.

[0129] In one embodiment, the homodimeric or heterodimeric protein is a homodimer of a monomer that is mouse IL12Rβ1, a mouse P40 subunit, an L1 linker, a mouse P35 subunit, an L1 linker, and a human IgG1, e.g., having the amino acid sequence set forth in SEQ ID No. 37.

[0130] In one embodiment, the homodimeric or heterodimeric protein is a monomeric homodimer of human IL12Rβ1, human P40 subunit, L1 linker, human P35 subunit, L1 linker, and human IgG1, e.g., having the amino acid sequence set forth in SEQ ID No. 38.

[0131] In one embodiment, the homodimeric or heterodimeric protein is a homodimer of a monomer that is mouse IL12Rβ2, a mouse P40 subunit, an L1 linker, a mouse P35 subunit, an L1 linker, and a human IgG1, e.g., having the amino acid sequence set forth in SEQ ID No. 39.

[0132] In one embodiment, the homodimeric or heterodimeric protein is a monomeric homodimer of human IL12Rβ2, human P40 subunit, L1 linker, human P35 subunit, L1 linker, and human IgG1, e.g., having the amino acid sequence set forth in SEQ ID No. 40.

[0133] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer which is a fusion protein of mouse IL12Rβ1, L2 linker, mouse P40 subunit, L1 linker, human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 41, and a second monomer which is a fusion protein of mouse IL12Rβ2, L2 linker, mouse P35 subunit, L1 linker, and human Fc-hole of IL12, e.g., having the amino acid sequence set forth in SEQ ID No. 43.

[0134] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer which is a fusion protein of human IL12Rβ1, an L2 linker, a human P40 subunit, an L1 linker, a human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 42, and a second monomer which is a fusion protein of human IL12Rβ2, an L2 linker, a human P35 subunit, an L1 linker, and a human Fc-hole of IL12, e.g., having the amino acid sequence set forth in SEQ ID No. 44.

[0135] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer which is a fusion protein of mouse IL12Rβ1, an L2 linker, a mouse P40 subunit, an L1 linker, a human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 45, and a second monomer which is a fusion protein comprising a mouse P35 subunit with an SP1 signal peptide, an L1 linker, and a human Fc-hole, e.g., having the amino acid sequence set forth in SEQ ID No. 47.

[0136] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer which is a fusion protein of human IL12Rβ1, an L2 linker, a human P40 subunit, an L1 linker, a human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 46, and a second monomer which is a fusion protein comprising a human P35 subunit with an SP1 signal peptide, an L1 linker, and a human Fc-hole, e.g., having the amino acid sequence set forth in SEQ ID No. 48.

[0137] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer: a fusion protein comprising a mouse P40 subunit, an L1 linker, and a human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 49, and a second monomer: a fusion protein comprising a mouse IL12Rβ2, an L2 linker, a mouse P35 subunit, an L1 linker, and a human Fc-hole, e.g., having the amino acid sequence structure set forth in SEQ ID No. 51.

[0138] In one embodiment, the homodimeric or heterodimeric protein is a heterodimer of a first monomer: a fusion protein comprising a human P40 subunit, an L1 linker, and a human Fc-knob, e.g., having the amino acid sequence set forth in SEQ ID No. 50, and a second monomer: a fusion protein comprising a human IL12Rβ2, an L2 linker, a human P35 subunit, an L1 linker, and a human Fc-hole, e.g., having the amino acid sequence structure set forth in SEQ ID No. 52.

[0139] In certain embodiments, the homodimeric or heterodimeric proteins are hydrolyzed by proteolytic enzymes that are specifically expressed in the tumor microenvironment.

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

[0141] In yet another aspect, the invention relates generally to polynucleotides encoding proteins, such as the fusion proteins or fragments thereof, disclosed herein.

[0142] In yet another aspect, the invention generally relates to expression vectors comprising a polynucleotide encoding a protein, such as a fusion protein or fragment disclosed herein.

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

[0144] In yet another aspect, the invention generally relates to a pharmaceutical composition comprising a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent.

[0145] In yet another aspect, the invention generally relates to a method for treating a disease or condition comprising administering to a patient a therapeutically effective amount of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof, as disclosed herein, wherein said disease or condition is selected from hyperplasia, solid tumors, or hematopoietic malignancies.

[0146] In certain embodiments, the disease or condition to be treated is hyperplasia.

[0147] In certain embodiments, the disease or condition to be treated is a solid tumor.

[0148] In certain embodiments, the disease or condition being treated is a hematopoietic malignancy.

[0149] In certain embodiments, the subject being treated is further administered one or more of chemotherapy, radiation therapy, targeted therapy, immunotherapy, or hormonal therapy.

[0150] In some embodiments, the method further comprises administering a chemotherapeutic agent to the subject.

[0151] In some embodiments, the method further comprises administering radiation therapy to the subject.

[0152] In some embodiments, the method further comprises administering a targeted therapy to the subject.

[0153] In some embodiments, the method further comprises administering immunotherapy to the subject.

[0154] In one embodiment, the method further comprises administering hormone therapy to the subject.

[0155] As used herein, the term "chemotherapeutic agent" refers to a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VERCADE®, Mremineium Pharm.), fulvestrant (FASLODEX®, AstraZeneca), stents (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith). kline), lonafarnib (SCH66336), sorafenib (BAY43-9006, Bayer Labs), and gefitinib (IRESA®, AstraZeneca), alkylating agents such as AG1478, AG1571 (SU5271; Sugen), thiotepa and CYTOXAN® cyclosphosphamide, alkyl sulfonates such as busulfan, improsulfan, and piposulfan, aziridines such as benzodopa, carboquone, metholedopa, and uredopa, ethylenimines and methylaminamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine, acetogens, antimycins (especially bullatacin and bullatacinone), camptothecins (including the synthetic analog topotecan), bryostatin, kallistatin, CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin), cryptophycins (especially cryptophycin 1 and cryptophycin 8), dolastatins, duocarmycins (including the synthetic analogs KW-2189, and CB1-TM1), eleutherobin, pancratistatin, sarcodictyin, spongiostatins, nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine,Antibiotics such as ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, nobembitine, phenesterine, prednimustine, trofosfamide, and uracil mustard, nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine, enediyne antibiotics (e.g., the calicheamicins, especially calicheamicin gammall and calicheamicin omegall (e.g., Agnew, Chem Intl. Ed. Engl., 33:183-186 (1994)), dynemicins including dynemicin A, bisphosphonates such as clodronate, esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, authramycin, azaserine, bleomycin, cactinomycin, carabimycin abicin), carminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin. anti-metabolites such as epirubicin, esonibicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, poporfiromycin, puromycin, quelamycin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin, methotrexate and 5-fluorouracil (5-FU), folic acid analogues such as denopterin, methotrexate, pteropterin, and trimetrexate, purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine, pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine,Carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine, androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone, antiadrenergics such as aminoglutethimide, mitotane, and trilostane, folic acid supplements such as frolinic acid, aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestrabucil, bisantrene, edatraxate, defofamine, demecolcine lcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucid, gallium nitrate, hydroxyurea, lentinan, lonidainine, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidanmol, nitraerine, pentostatin, phenamet, pirarubicin, losoxantrone, 2-ethylhydrazide, procarbazine, PSK® polysaccharide complex (JHS Natural Products, Eugene, OR), razoxane, rhizoxin, sizofiran, spirogermanium, tenuazonic acid, triaziquone, 2,2',2"-trichlorotriethylamine, trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine), urethane, vindesine, dacarbazine, mannomustine, mitobronitol, mitolactol, pipobroman, gacytosine, arabinoside ("Ara-C"), cyclophosphamide, thiotepa, taxoids such as TAXOL® (paclitaxel, Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® (cremophor free),Albumin engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE® doxetaxel, Rhone-Poulenc Rorer, Antony, France), chloranbucil, GEMZAR® (gemcitabine), 6-thioguanine, mercaptopurine, methotrexate, platinum analogs such as cisplatin and carboplatin, vinblastine, etoposide (VP-16), ifosfamide, mitoxantrone, vincristine, NAVELBINE® (vinorelbine), novantrone, teniposide, edatrexate, daunomycin, aminopterin, capecitabine (XELODA®), ibandronate, CPT-11, topoisomerase inhibitors, RFS2000, difluoromethylomithine (DMFO), retinoids such as retinoic acid, and pharmaceutically acceptable salts, acids, and derivatives of any of the above.

[0156] Examples of second (or further) agents or therapeutic agents include immunotherapeutic agents (e.g., PD-1 inhibitors (pembrolizumab, nivolumab, cemiplimab), PD-L1 inhibitors (atezolizumab, avelumab, durvalumab), CTLA4 antagonists (ipilimumab), cell signaling inhibitors (e.g., imatinib, gefitinib, bortezomib, erlotinib, sorafenib, sunitinib, dasatinib, vorinostat, lapatinib, temsirolimus, nilotinib, everolimus, pazopanib, trastuzumab, bevacizumab, cetuximab, ranibizumab, pegaptanib, panitumumab, etc.), mitotic inhibitors agents (e.g., paclitaxel, vincristine, vinblastine, etc.), alkylating agents (e.g., cisplatin, cyclophosphamide, clomabucil, carmustine, etc.), antimetabolites (e.g., methotrexate, 5-FU, etc.), insertional anticancer agents (e.g., actinomycin, anthracyclines, bleomycin, mitomycin-C, etc.), topoisomerase inhibitors (e.g., irinotecan, topotecan, teniposide, etc.), immunotherapeutic agents (e.g., interleukins, interferons, etc.), and antihormonal agents (e.g., tamoxifen, raloxifene, etc.).

[0157] In yet another aspect, the invention generally relates to the use of a protein, such as a fusion protein or fragment thereof, disclosed herein, to treat or ameliorate a disease or disorder, such as a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0158] In yet another aspect, the invention generally relates to the use of polynucleotides encoding proteins, such as the fusion proteins disclosed herein, or fragments thereof, to treat or ameliorate a disease or disorder, such as a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0159] In another aspect, the invention generally relates to the use of a protein, such as a fusion protein or fragment thereof disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or ameliorating a disease or disorder, such as a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0160] In yet another aspect, the invention generally relates to the use of a polynucleotide encoding a protein, such as a fusion protein or fragment thereof disclosed herein, and a pharma- ceutically acceptable excipient, carrier, or diluent in the preparation of a medicament for treating or ameliorating a disease or disorder, such as a hyperplasia, a solid tumor, or a hematopoietic malignancy.

[0161] In one embodiment, the drug is an anti-cancer drug.

[0162] In one embodiment, the disease or disorder is one or more selected from head and neck cancer, endometrial cancer, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, renal cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain cancer, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

[0163] In certain embodiments, the anti-cancer agent is effective in treating B cell lymphoma or anti-colon cancer.

[0164] In yet another aspect, the invention generally relates to cell lines comprising polynucleotides encoding proteins, such as the fusion proteins or fragments disclosed herein.

[0165] In another aspect, the invention generally relates to a method for producing a protein, the method comprising culturing said cell line, hi one embodiment, the method further comprises purifying or isolating the resulting protein, such as a fusion protein or fragment thereof disclosed herein.

[0166] In yet another aspect, the invention generally relates to a method for producing a protein, the method comprising the steps of providing an expression vector encoding a protein, such as a fusion protein or fragment thereof as disclosed herein, introducing the expression vector into a host cell, culturing the host cell in a medium under conditions sufficient to express the protein, and purifying the protein from the host cell or medium.

[0167] Any suitable expression vector may be utilized. An exemplary expression vector is the pEE12.4 expression vector.

[0168] Any suitable host cell may be utilized, such as 293F cells or CHO cells.

[0169] Introduction of the expression vector can be achieved by any suitable transfection method, and can be performed via transient transfection or stable cell lines.

[0170] Any suitable purification method may be utilized, an exemplary purification method is by affinity chromatography with Protein A / G, or by size exclusion methods.

[0171] In another aspect, the invention relates generally to isolated proteins produced by the methods disclosed herein.

[0172] In some embodiments, the isolated protein is substantially pure.

[0173] As disclosed herein, linker sequences can be used to join two or more polypeptides of a biologically active polypeptide to generate a single chain molecule with a desired functional activity.

[0174] Any suitable linker may be utilized. Exemplary peptide linker sequences include sequences having about 7-20 amino acids, such as about 8-16 amino acids. The linker sequence is preferably flexible so as not to hold the biologically active polypeptide or effector molecule in one undesired conformation. For example, the linker sequence can be used to space the recognition site and the fusion molecule. Specifically, the peptide linker sequence can be positioned to provide molecular flexibility. The linker preferably includes primarily amino acids with small side chains, such as glycine, alanine, and serine, to provide flexibility.

[0175] In general, the preparation of the fusion protein complex of the present invention can be accomplished by procedures disclosed herein and by recognized recombinant DNA techniques, including, for example, polymerase chain amplification reaction (PCR), preparation of plasmid DNA, cleavage of DNA with restriction enzymes, preparation of oligonucleotides, ligation of DNA, isolation of mRNA, introduction of DNA into appropriate cells, transformation or transfection of the host, and culture of the host.Furthermore, the fusion molecule can be isolated and purified using chaotropic agents and well-known electrophoretic, centrifugation, and chromatographic 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) for disclosures regarding these methods).

[0176] The present invention further provides nucleic acid and DNA sequences encoding the fusion proteins. The DNA sequences may be carried by vectors suitable for extrachromosomal replication, such as phages, viruses, plasmids, phagemids, cosmids, YACs, or episomes. For example, DNA vectors encoding the desired fusion proteins may be used to facilitate the preparation methods described herein and to obtain substantial quantities of the fusion proteins or components thereof. The DNA sequences may be inserted into a suitable expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. A variety of host-vector systems may be utilized to express the protein coding sequence. These systems include mammalian cell systems infected with viruses (e.g., vaccinia virus, adenovirus, etc.), insect cell systems infected with viruses (e.g., baculovirus), microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used (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) for disclosures regarding these methods).

[0177] The fusion protein components encoded by the DNA vector can be provided in a cassette format. The term "cassette" means that each component can be easily replaced with another by standard recombinant methods. In particular, DNA vectors configured in a cassette format are particularly desirable when the encoded fusion complex is used against a pathogen that has or may have the ability to generate serotypes.

[0178] To create a vector encoding a fusion protein complex, the sequence encoding the biologically active polypeptide is ligated to the effector peptide using an appropriate ligase. DNA encoding the present peptide can be obtained by isolating DNA from a natural source, such as a suitable cell line, or by known synthetic methods, such as the phosphotriester method (Oligonucleotide Synthesis, IRL Press, MJ Gait, ed., 1984). Synthetic oligonucleotides may be prepared using commercially available automated oligonucleotide synthesizers. Once isolated, the gene encoding the biologically active polypeptide can be amplified by PCR or other means known in the art. Suitable PCR primers for amplifying the biologically active polypeptide gene can add restriction sites to the PCR product. The PCR product preferably contains splice sites for the effector peptide and leader sequences necessary for proper expression and secretion of the biologically active polypeptide-effector fusion complex. The PCR product also preferably contains sequences encoding linker sequences, or restriction enzyme sites for ligation of such sequences.

[0179] The fusion proteins described herein may be produced by standard recombinant DNA techniques. For example, once a DNA molecule encoding a biologically active polypeptide is isolated, the sequence may be joined to another DNA molecule encoding an effector polypeptide. The nucleotide sequence encoding the biologically active polypeptide may be directly joined to the DNA sequence encoding the effector peptide, or more commonly, a DNA sequence encoding a linker sequence as discussed herein may be inserted between the sequence encoding the biologically active polypeptide and the sequence encoding the effector peptide and joined using a suitable ligase. The resulting hybrid DNA molecule may be expressed in a suitable host cell to produce a fusion protein complex. The DNA molecules are ligated to each other in a 5' to 3' orientation such that, after ligation, the translation frame of the encoded polypeptide is not altered (i.e., the DNA molecules are ligated to each other in frame). The resulting DNA molecule encodes an in-frame fusion protein.

[0180] Other nucleotide sequences can also be included in the genetic construct. For example, a promoter sequence that controls the expression of a sequence encoding a biologically active polypeptide fused to an effector peptide, or a leader sequence that directs the fusion protein to the cell surface or to the culture medium, can be included in the construct or can be present in an expression vector into which the construct is inserted.

[0181] In obtaining a mutant biologically active polypeptide, IL12, IL12R, or Fc domain coding sequence, those skilled in the art will recognize that the polypeptide may be modified by certain amino acid substitutions, additions, deletions, and post-translational modifications without loss or reduction of biological activity. In particular, it is well known that conservative amino acid substitutions, i.e., the substitution of one amino acid for another amino acid with similar size, charge, polarity, and conformation, are unlikely to significantly modify protein function. The 20 standard amino acids that are the building blocks of proteins can be broadly classified into four groups of conservative amino acids as follows: Non-polar (hydrophobic) groups include alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine; polar (uncharged, neutral) groups include asparagine, cysteine, glutamine, glycine, serine, threonine, and tyrosine; positively charged (basic) groups include arginine, histidine, and lysine; and negatively charged (acidic) groups include aspartic acid and glutamic acid. Substitution of one amino acid in a protein for another within the same group is unlikely to adversely affect the biological activity of the protein. In other examples, modifications to amino acid positions can be made to reduce or enhance the biological activity of a protein. Such changes can be introduced randomly or via site-directed mutagenesis based on known or predicted structural or functional properties of the target residue. After expression of the mutant protein, changes in biological activity due to modifications can be readily assessed using binding or functional assays.

[0182] Homology between nucleotide sequences can be determined by DNA hybridization analysis, in which the stability of double-stranded DNA hybrids depends on the degree of base pairing that occurs. Conditions of high temperature and / or low salt content decrease the stability of these hybrids, and the conditions can be varied to prevent annealing of sequences with less than the selected homology. For example, for a sequence with a GC content of about 55%, hybridization and washing conditions of 40-50C, 6x SSC (sodium chloride / sodium citrate buffer), and 0.1% SDS (sodium dodecyl sulfate) will indicate about 60-70% homology, hybridization and washing conditions of 50-65C will indicate about 82-97% homology, and hybridization and washing conditions of 52C, 0.1x SSC, and 0.1% SDS will indicate about 99-100% homology. A wide variety of computer programs are also available for comparing nucleotide to amino acid sequences (and measuring the degree of homology). Readily available sequence comparison and multiple sequence alignment algorithms are the Basic Local Alignment Search Tool (BLAST) and ClustalW programs, respectively.

[0183] A number of strategies can be utilized to express the protein fusion complexes of the invention. For example, the fusion protein constructs described above can be incorporated into an appropriate vector by known means, such as the use of restriction enzymes, to cleave the vector for insertion of the construct followed by ligation. The vector containing the genetic construct is then introduced into an appropriate host to express the fusion protein (see Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed. (1989) for disclosure regarding these methods).

[0184] Selection of an appropriate vector can be made empirically based on factors related to the cloning protocol. For example, the vector must be compatible with and carry a suitable replicon for the host to be used. Furthermore, the vector must be compatible with the DNA sequence encoding the fusion protein complex to be expressed. Suitable host cells include eukaryotic and prokaryotic cells, preferably cells that are easily transformable and exhibit rapid growth in culture medium. In particular, preferred host cells include prokaryotes such as E. coli, Bacillus subtillus, and eukaryotes such as animal cells and yeast strains, for example S. cerevisiae. Mammalian cells in general are preferred, particularly J558, NSO, SP2-O, or CHO. Other suitable hosts include insect cells, for example Sf9. Conventional culture conditions are utilized. See 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, expression of the fusion protein complex bound to an immunoglobulin can be determined by ELISA specific for the bound immunoglobulin and / or by immunoblotting. Other methods for detecting expression of a fusion protein comprising a biologically active polypeptide bound to an IL12 or IL12R domain are disclosed in the Examples.

[0185] Host cells can be used for preparative purposes to propagate nucleic acids encoding the desired fusion protein or components thereof. Host cells can include prokaryotic or eukaryotic cells for which production of the fusion protein is specifically intended. Host cells thus specifically include yeast, fly, worm, plant, frog, mammalian cells, and organisms capable of propagating nucleic acids encoding the fusion. Non-limiting examples of mammalian cell lines that can be used include CHO dhfr cells (Urlaub and Chasm, 1980 Proc. Natl. Acad. Sci. USA, 77:4216), 293 cells (Graham et al. 1977 J. Gen. Virol., 36:59()), or myeloma cells such as SP2 or NSO (Galfre and Milstein, 1981 Meth. Enzymol., 73(B):3).

[0186] Host cells capable of propagating nucleic acids encoding the desired fusion protein complexes also include non-mammalian eukaryotic cells, including insect (e.g., Sp. frugiperda), yeast (e.g., S. cerevisiae, S. pombe, P. pastoris, K. lactis, H. polymorpha, generally as reviewed in Fleer, R., 1992 Current Opinion in Biotechnology, 3(5):486-496), fungi, and plant cells. Certain prokaryotes, such as E. coli and Bacillus, are also contemplated.

[0187] Nucleic acids encoding the desired fusion proteins can be introduced into host cells by standard techniques for transfecting cells. The term "transfecting" or "transfection" is intended to encompass calcium phosphate co-precipitation, DEAE-dextran mediated transfection, lipofection, electroporation, microinjection, viral transduction, and / or integration.

[0188] Various promoters (transcription initiation regulatory regions) may be used according to the present invention. The selection of an appropriate promoter depends on the proposed expression host. Promoters from heterologous sources may be used as long as they are functional in the selected host.

[0189] The choice of promoter also depends on the desired efficiency and level of peptide or protein production. Inducible promoters, such as tac, are often utilized to dramatically increase protein expression levels in E. coli. Overexpression of a protein can be harmful to the host cell. As a result, host cell growth can be limited. Use of an inducible promoter system allows the host cells to be cultured to an acceptable density before inducing gene expression, facilitating greater production.

[0190] A variety of signal sequences may be used in accordance with the present invention. A signal sequence that is homologous to the biologically active polypeptide coding sequence may be used. Alternatively, a signal sequence selected or designed for efficient secretion and processing in the expression host may be used. The signal sequence may be directly linked to the protein coding sequence via a sequence encoding a signal peptidase cleavage site or may be linked by a short nucleotide bridge.

[0191] Expression constructs can be assembled using known recombinant DNA techniques. Restriction enzyme digestion and ligation are the basic steps used to join two fragments of DNA. Polylinkers and adapters may be used to facilitate joining of selected fragments. The expression constructs are typically assembled stepwise using multiple rounds of restriction, ligation, and transformation of E. coli. A number of cloning vectors suitable for constructing expression constructs are known in the art (λZAP and pBLUESCRIPT SK-1, Stratagene, La Jolla, Calif., pET, Novagen Inc., Madison, Wis.).

[0192] The expression construct may be transformed into the host as either a linear or circular cloning vector construct, or may be removed from the cloning vector and then 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 of a number of known gene transfer systems (e.g., natural competence, chemically mediated transformation, protoplast transformation, electroporation, biological transformation, transfection, or conjugation). The gene transfer system selected depends on the host cell and vector system used.

[0193] The present invention further provides a manufacturing process for isolating a fusion protein of interest. In this process, host cells (e.g., yeast, fungal, insect, bacterial, or animal cells) that have been introduced with a nucleic acid encoding a protein of interest operably linked to a regulatory sequence are grown at a production scale in a culture medium to stimulate transcription of the nucleotide sequence encoding the fusion protein of interest. The fusion protein of interest is then isolated from the harvested host cells or from the culture medium. Standard protein purification techniques can be used to isolate the protein of interest from the medium or the harvested cells. In particular, purification techniques can be used to express and purify the desired fusion protein on a large scale (i.e., at least milligram quantities) from various embodiments including roller bottles, spinner flasks, tissue culture plates, bioreactors, or fermenters.

[0194] The expressed protein fusion complex can be isolated and purified by known methods. Typically, the culture medium is centrifuged or filtered, and the supernatant is then purified by affinity or immunoaffinity chromatography, such as Protein-A or Protein-G affinity chromatography, or immunoaffinity protocols including the use of monoclonal antibodies that bind to the expressed fusion complex, such as the bound TCR or immunoglobulin regions thereof. The fusion proteins of the present invention can be isolated and purified by an appropriate combination of known techniques. These methods include, for example, methods that use solubility, such as salt precipitation and solvent precipitation; methods that use molecular weight differences, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide gel electrophoresis; methods that use charge differences, such as ion exchange column chromatography; methods that use specific affinities, such as affinity chromatography; methods that use hydrophobicity differences, such as reversed-phase high performance liquid chromatography; methods that use isoelectric point differences, such as isoelectric focusing; and metal affinity columns, such as Ni-NTA (for disclosures regarding 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)).

[0195] The fusion proteins of the present invention are preferably substantially pure; that is, the fusion protein is isolated from the cellular constituents that normally accompany it, so that the fusion protein is preferably present in at least 80% or 90%-95% homogeneity (w / w). Fusion proteins having at least 98%-99% homogeneity (w / w) are most preferred for many pharmaceutical, clinical, and research applications. Once substantially purified, the fusion protein must be rendered substantially free of contaminants for therapeutic use. Once partially purified to substantial purity, the soluble fusion protein can be used therapeutically or to perform the in vitro or in vivo assays disclosed herein. Substantial purity can be determined by a variety of standard techniques, such as chromatography and gel electrophoresis.

[0196] The invention also provides a pharmaceutical preparation comprising a therapeutically effective amount of a composition, fusion protein, polynucleotide, genetic construct, vector, or host cell according to the invention and a pharma- ceutically acceptable excipient or vehicle.

[0197] Preferred excipients for use in the present invention include sugars, starches, celluloses, gums, and proteins. In a preferred embodiment, the pharmaceutical compositions of the present invention are formulated in pharmaceutical forms for administration as solids (e.g., tablets, capsules, lozenges, granules, suppositories, crystalline or amorphous sterile solids capable of being reconstituted to provide a liquid form, etc.), liquids (e.g., solutions, suspensions, emulsions, elixirs, lotions, unguents, etc.), or semisolids (gels, ointments, creams, etc.). The pharmaceutical compositions of the present invention can be administered by any route, including, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracerebroventricular, transdermal, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal routes. A revision of the different forms of administration of the active principle, the excipients used, and the procedures for their preparation can be found in Remington's Pharmaceutical Sciences (AR Gennaro, Ed.), 2001, pp. 117-119, 1997. thedition, Williams & Wilkins PA, USA (2000). Examples of pharma- ceutically acceptable vehicles are known in the art and include phosphate-buffered saline, water, emulsions such as oil / water emulsions, different types of moisturizing agents, sterile solutions, etc. Compositions containing said vehicles can be formulated by conventional procedures known in the art.

[0198] In the case of the pharmaceutical composition of the present invention that contains a nucleic acid (polynucleotide, vector, or gene construct of the present invention), the present invention contemplates a pharmaceutical composition that is specially prepared for administering said nucleic acid. This pharmaceutical composition can contain said nucleic acid in naked form, that is, without compounds that protect the nucleic acid from degradation by biological nucleases. This has the advantage of eliminating the toxicity associated with the reagents used for transfection. Suitable administration routes for naked compounds include intravascular, intratumoral, intracranial, intraperitoneal, intrasplenic, intramuscular, subretinal, subcutaneous, mucous, topical, and oral routes (Templeton, 2002 DNA Cell Biol., 21:857-867). Alternatively, the nucleic acid may be administered as part of a liposome, conjugated to cholesterol, or conjugated to a compound that allows for enhanced translocation across a cell membrane, such as the Tat peptide 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 such as those described in WO07069090 (Lindgren, et al. 2000 Trends Pharmacol. Sci 21:99-103; Schwarze, et al. 2000 Trends Pharmacol. Sci. 21:45-48; Lundberg, et al. 2003 Mol. Therapy 8:143-150, and Snyder, et al. 2004 Pharm. Res. 21:389-393). Alternatively, the polynucleotide may be administered which forms part of a plasmid vector or a viral vector, preferably a vector based on an adenovirus, an adeno-associated virus or a retrovirus, such as a vector based on a murine leukemia virus (MLV) or a lentivirus (HIV, FIV, EIAV).

[0199] The compositions of the invention contain 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 drug, i.e., about 4.4×10 16 The single dose can be administered at a dose of 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, or 0.075 nmol per copy or kg of body weight. The single dose can be administered by injection, inhalation, or topical administration. The bifunctional polynucleotides and compositions of the invention can be administered directly to an organ in which the target mRNA is expressed, in which case the dosage will be administered at 0.00001 mg to 3 mg per organ, preferably 0.0001 to 0.001 mg per organ, about 0.03 to 3.0 mg per organ, about 0.1 to 3.0 mg per organ, or 0.3 to 3.0 mg per organ.

[0200] The dosage depends on the severity of the condition being treated and the response thereto, and may vary from several days to several months, or until remission of the condition is observed. The optimal dosage can be determined by periodically measuring the drug concentration in the patient's body. The optimal dosage can be determined from EC50 values ​​obtained from previous in vitro or in vivo testing on animal models. The unit dosage can be administered once a day, or less than once a day, preferably less than once every 2, 4, 8, or 30 days. Alternatively, a single initial dose can be administered, followed by one or more maintenance doses, usually in amounts less than the initial dose. This maintenance regime may require treating the patient with a dosage of 0.01 μg to 1.4 mg per kg of body weight per day, for example 1, 0.1, 0.01, 0.001, or 0.00001 mg / kg of body weight per day. The maintenance dose is preferably administered at most once every 5, 10, or 30 days. This treatment must be continued for a period of time that varies depending on the type of changes the patient undergoes, their severity, and the condition of the patient. After treatment, the patient's evolution must be monitored to determine whether the dosage should be increased if the disease does not respond to treatment, or whether the dosage should be decreased if improvement in the disease or undesirable secondary effects is observed.

[0201] Depending on the particular situation, the daily dosage can be administered in a single dose, or in two or more doses. If repeated or frequent administration is required, implantation of an administration device such as a pump, long-term catheter (intravenous, intraperitoneal, intrathoracic, or intracapsular), or reservoir is recommended.

[0202] The compositions of the present invention may be administered according to methods known to those of skill in the art, including, but not limited to, intravenous, oral, nasal, parenteral, topical, transdermal, rectal, and the like.

[0203] The following examples are meant to illustrate the practice of the present invention but are not meant to limit it in any way. EXAMPLES

[0204] The following examples illustrate certain exemplary embodiments of compounds prepared according to the disclosed invention. It will be understood that the following general methods, and other methods known to those skilled in the art, are applicable to the compounds disclosed herein and to subclasses and species thereof.

[0205] Example 1. Design of seven IL12-Fc prodrugs IL12 has two subunits, p35 and p40. The Fc segment of human IgG1, human Fc-knob, and human Fc-hole were used to construct the corresponding prodrugs. The prodrug designs combine the two subunits of IL12 in series or parallel. The specific forms are as follows:

[0206] FIG. 1 is a schematic showing the structure of homodimeric IL12-Fc (Homo IL12), in which two subunits are linked in tandem and the molecular weight of the dimer is 175 KD (MW=175 KD).

[0207] Figure 2 is a schematic diagram showing the structure of heterodimeric IL12-Fc (Het IL12), in which two subunits are linked in parallel. The molecular weight of the dimer is 115 KD (MW=115 KD), and the P35 and P40 signal peptides were added to the N-terminus of the P35 and P40 subunits, respectively.

[0208] The prodrug forms block IL12 from binding to either of its receptors or to both receptors simultaneously (IL12Rβ1 and IL12Rβ2). A portion of the extracellular domain of IL12Rβ1, consisting of two fibronectin type-III domains (I+II) at the N-terminus of IL12Rβ1, called Rβ1, is fused to the N-terminus of P40. Rβ1 acts as a decoy that competitively prevents endogenous IL12Rβ1 interaction with P40. P35 is blocked using two fibronectin type-III domains (I+II) at the N-terminus of IL12Rβ2, called Rβ2, thereby preventing P35 interaction with endogenous IL12Rβ2.

[0209] The prodrug construct for homodimer-IL12 is shown in FIG.

[0210] FIG. 3 is a schematic diagram of the prodrug structure of homodimeric IL12Rβ1 (referred to as Homo-R1), with a dimer MW=232 KD.

[0211] FIG. 4 is a schematic diagram of the prodrug structure of homodimeric IL12Rβ2 (referred to as Homo-R2), with a dimer MW=250 KD.

[0212] FIG. 5 is a schematic diagram of the prodrug structure of the heterodimer IL12Rβ1 / Rβ2 (referred to as Het-R1 / R2), dimer MW=182 KD.

[0213] FIG. 6 is a schematic diagram of the prodrug structure of heterodimeric IL12Rβ1 (referred to as Het-R1), a dimer MW=144 KD in which the P35 signal peptide was added to the C-terminus of the fusion segment P35 of the P35-Fc-hole monomer.

[0214] FIG. 7 is a schematic diagram of the prodrug structure of heterodimeric IL12Rβ2 (referred to as Het-R2), a dimer MW=153 KD in which the P40 signal peptide was added to the C-terminus of the fusion segment P40 of the P40-Fc-knob monomer.

[0215] Example 2. Structure, purification, and production of IL12 prodrugs The seven proteins described in Example 1 were expressed and produced. First, the recombinant DNA of each fusion protein constructed on the expression vector pEE12.4 was transfected into 293F or CHO cells. The host cells were cultured and the cell supernatant was collected. Then, the protein was purified from the supernatant by a protein A / G affinity chromatography column.

[0216] For the expression of homodimeric proteins, the constructed expression vector plasmid was transferred into 293 or CHO cell host. The plasmid expression product of the cells can spontaneously form homodimeric proteins. For the expression of heterodimeric proteins, it is necessary to transfect with two expression vector plasmids with the same molar ratio, and the monomers expressed in the cells can also spontaneously form heterodimers.

[0217] The results of SDS-PAGE electrophoresis are shown in Figures 8A and 8B.

[0218] The procedures used to construct the vectors, transfect the host cells, and induce expression are described below.

[0219] Expression plasmids for various fusion proteins were constructed on the PEE12.4 vector and transfected into 293F or CHO cells. The proteins expressed in the culture supernatant were purified by protein A affinity chromatography column.

[0220] The expression vector was constructed as follows: (1) PEE12.4-HindIII-p40 (signal)-NruI-p35 (no signal)-BsiWI-hIgG1-EcoRI (2) PEE12.4-HindIII-P35(signal)-BsiWI-Fch-EcoRI (3) PEE12.4-HindIII-P40(signal)-NruI-Fck-EcoRI (4) PEE12.4-HindIII-IL12Rb1 -BsiWI-p40 (no signal)-NruI-p35 (no signal)-BsiWI-hIgG1-EcoRI (5) PEE12.4-HindIII-IL12Rb2-BsiWI-p40 (no signal)-NruI-p35 (no signal)-BsiWI-hIgG1-EcoRI (6) PEE12.4-HindIII-IL12Rb1-BsiWI-p40 (no signal)-NruI-Fck-EcoRI (7) PEE12.4-HindIII-IL12Rb2-BstBI-p35(no signal)-BsiWI-Fch-EcoRI.

[0221] HindIII, NruI, BsiWI, and EcoRI are enzyme cleavage sites.

[0222] The linkage sequences between each fusion protein segment are as follows: (1) Homo IL12: linker L1 between P40 and P35, linker L1 between P35 and Fc. (2) Het IL12: linker L1 between P40 and Fc, linker L1 between P35 and Fc. (3) Linker L2 between Rβ1 and P40; the corresponding target sequence for the protease is SGRSENIRTA. (4) Linker L2 between Rβ2 and P40; the corresponding protease target sequence is SGRSENIRTA. (5) Linker L2 between Rβ2 and P35; the corresponding target sequence for the protease is SGRSENIRTA.

[0223] Transfection can be performed stably or transiently. The protocol for transient transfection is as follows: (1) Cell resuscitation: Free 293F cells were resuscitated at 3 × 10 7The cells were cryopreserved in CD OptiCHO™ medium (containing 10% DMSO) at a concentration of 1000 cells / mL. After removal from liquid nitrogen, they were quickly thawed in a 37°C water bath, added to a 15 mL centrifuge tube containing 10 mL of OptiCHO™ medium, and centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded, and the cell pellet was suspended and cultured in 30 ml of OptiCHO™ medium at 37°C, 8% CO2, and 135 rpm. After 4 days, the cells were expanded and cultured at an expanded concentration of 3 x 10 6 cells / mL or less. (2) Two days before transfection, 0.6-0.8x10 cells were cultured in suspension using 293F cells. 6 Transient transfections (200 ml) were performed at a seeding density of cells / ml. (3) After 2 days, the transfected cell suspension was counted (2.5-3.5 × 10 6 cells / mL) and the expected cell density, the cell suspension was centrifuged at 1,000 rpm for 5 min and the supernatant was discarded. (4) The cells were resuspended in 50 mL of fresh free 293 medium, centrifuged again at 1,000 rpm for 5 minutes, and the supernatant was discarded. (5) The 293F cells were resuspended in 200 mL of free-form 293 medium. (6) 600 μg of plasmid was diluted in 5 mL of free-form 293 medium and sterilized by filtration using a 0.22 μM filter. (7) 1.8 mg of PEI was diluted with 5 mL of free 293 medium and sterilized by filtration using a 0.22 μM filter. Immediately, 5 mL of plasmid and 5 mL of PEI were mixed and left to stand at room temperature for 5 minutes. (8) The plasmid / PEI mixture was added to the cell suspension and placed in an incubator at 37°C, 8% CO2, 85 rpm, and supplemented with 50 μg / L LONG™ R3 IGF-1 growth factor. (9) After 4 hours, 200 ml of EX-CELL™ 293 medium and 2 mM glutamine were added, and the rpm was adjusted to 135 to continue culturing. (10) After 24 hours, 3.8 mM VPA, a cell proliferation inhibitor, was added. Supernatants were collected between 4 and 8 days post-transfection (ensure that cell viability was >70% when collected for optimal protein quality).

[0224] Fusion protein harvest, purification, and electrophoretic validation: (1) Sample preparation: The suspension cell culture solution was transferred to a 500 mL centrifuge barrel and centrifuged at 8,000 rpm for 20 min. The supernatant was discarded and impurities were removed using a 0.45 μM filter. 0.05% NaN3 was added to prevent bacterial growth during the purification process. (2) Column assembly: An appropriate amount of Protein A agarose was added to the column (20 mg of human Fc fusion protein per mL of Protein A) and incubated with 20% ethanol solution at room temperature for about 10 minutes. The outlet of the column was opened to slowly release the ethanol solution by gravity. (3) The column was washed and equilibrated with 10 column volumes of distilled water and binding buffer (20 mM sodium phosphate + 0.15 M NaCl, pH 7.0), respectively. (4) The sample was pumped continuously through the column at a flow rate of 10 column volumes per hour. (5) The column was washed with 10 column volumes of binding buffer until no protein was detected in the flow-through. (6) Elution buffer (0.1 M glycine, pH 2.7) was used for elution, and the eluate was collected in 1 mL units. An appropriate amount of 1 M Tris (pH 9.0) was added to neutralize (pH should be adjusted to 6-8, and the isoelectric point of the purified protein should be 0.5 or higher). (7) Using Zeba desalting or concentration spin columns, the target protein solution was exchanged into the desired buffer (adjusting the pH of the buffer to avoid the isoelectric point of the protein). Protein concentration was determined by SDS-PAGE electrophoresis (2.5 μg protein loading per sample) and NanoDrop2000 using BSA as a standard.

[0225] After elution, the column was washed successively with 20 column volumes of distilled water, then the column was washed with 10 column volumes of 20% ethanol. Finally, the ethanol solution was immersed into the gel medium and stored at 4°C.

[0226] Example 3. In vivo antitumor activity of fusion proteins Systemic injection of IL12-Fc completely eliminated MC38 tumors, with Het IL12 being more potent than Homo IL12. To determine whether IL12-Fc can effectively eliminate tumors during systemic administration and to compare the therapeutic effects of both forms of IL12-Fc, the MC38 mouse model was used in mice, and cohorts were treated with various doses by systemic administration. The results of the study are shown in Figure 9.

[0227] FIG. 9A: On day 0, WT C57BL / 6 mice (n=5 / group) were administered 5×10 5 MC38 cells were inoculated subcutaneously. Tumor volumes of cancer-bearing mice were recorded on days 13, 16, and 20 by intraperitoneal injection of PBS, 0.5 μg, 1 μg, 5 μg, and 10 μg of Homo IL12.

[0228] FIG. 9B: On day 0, WT C57BL / 6 mice (n=5 / group) were administered 5×10 5 MC38 cells were inoculated subcutaneously. Tumor volumes of cancer-bearing mice were recorded on days 13, 16, and 20 by intraperitoneal injection of PBS, 0.5 μg, 1 μg, 5 μg, and 10 μg of Homo IL12.

[0229] These results indicate that IL12-Fc is effective in eliminating tumors, with Het IL12 being more potent than Homo IL12. Systemic use of IL12-Fc causes serious side effects, and Het IL12 is more toxic than Homo IL12.

[0230] Because IL12 receptors are widely present in T, B, and NK cells, the use of IL12 is often accompanied by strong toxic side effects. Clinically, patients are mainly characterized by various blood disorders and liver toxicity. Various inflammatory cytokines in mouse serum were experimentally measured as a major indicator of toxicity caused by IL12-Fc.

[0231] On day 0, 5x10 5 MC38 cells were inoculated subcutaneously into WT C57BL / 6 mice (n=5 / group); PBS, 5 μg Homo IL12 or Het IL12 were injected intraperitoneally on days 13, 16, and 20. Six hours after administration on day 20, blood was collected from the ocular vessels to detect the levels of inflammatory factors IL12p70, IFN-γ, TNF, MCP-1, IL-10, and IL-6 in the serum.

[0232] As a result, both Homo IL12 and Het IL12 induced potent cytotoxicity, with Het IL12 being more cytotoxic than Homo IL12, as shown in Figure 10. Het IL12 receptor prodrug effectively eliminates MC38 tumors.

[0233] In order to weaken the decoy power of IL12 in the tumor microenvironment, IL12 prodrugs were constructed by binding IL12 decoy receptors using substrate sequences that are sensitive to certain proteases. Proteases that can cleave substrates are more highly expressed in certain tumor types compared to normal tissues, thus increasing the localization of active IL12 at tumor sites while decreasing the systemic toxicity of IL12. In in vivo experiments, Het IL12 produced more potent antitumor effects and was more toxic against MC38 tumors compared to Homo IL12 when doses of less than 5 μg / mouse were used. To test the concept of prodrugs as a means to reduce the toxicity of IL12 treatment in vivo, Het IL12 (described in Example 1) was bound to decoy IL12 receptors in several configurations (e.g., Het-R1, Het-R2, Het-R1 / R2). Homo IL12 bound to decoy IL12 receptors (eg, Homo-R1, Homo-R2) was also tested.

[0234] Figure 11A: 5x10 on day 0 5 MC38 cells were inoculated subcutaneously into WT C57BL / 6 mice (n=5 / group) and on days 10, 13, and 16, 5 μg Het IL12, Het-R1, Het-R2, or Het-R1 / R2 were inoculated subcutaneously into WT C57BL / 6 mice (n=5 / group), control groups were treated with PBS.

[0235] Figure 11B: On day 0, 5x10 5 MC38 cells were inoculated subcutaneously into WT C57BL / 6 mice (n=5 / group). On days 10, 13, and 16, 2.5 μg of Het IL12, Het-R1, Het-R2, or Het-R1 / R2 were injected intraperitoneally, and the control group was treated with PBS.

[0236] The results showed that the three forms of Het IL12 prodrugs were effective in eliminating tumors. At a dose of 5 μg / mouse, the three forms of Het IL12 prodrugs had better antitumor effects against MC38 compared with Homo IL12, and even when the dose was reduced to 2.5 μg / mouse, the Het IL12 prodrugs (Het R1, Het R2, or Het R1 / R2) could still effectively control tumors. The Het IL12 prodrugs bound to the IL12 receptor had fewer side effects when administered systemically.

[0237] The body weight changes of mice were recorded after systemic administration of different drugs / prodrugs and the expression levels of inflammatory cytokines in serum were collected from the ocular blood vessels of mice.

[0238] Figure 12A: 5x10 on day 0 5 WT C57BL / 6 mice (n=5 / group) were subcutaneously inoculated with 1000 mg of MC38 cells and 5 μg of Het IL12, Het-R1, Het-R2, or Het-R1 / R2 on days 10, 13, and 16, and the control group was treated with PBS. Mice were weighed during treatment.

[0239] Figure 12B-G: Blood was obtained from the ocular vessels of mice during treatment. Serum levels of the inflammatory cytokines IL12p70, TNF, IFN-γ, MCP-1, IL-10, and IL-6 were measured.

[0240] The results showed that Het IL12 conjugated to IL12 receptor prodrugs (Het-R1, Het-R2) had less toxic side effects compared to Het IL12 not conjugated to the IL12 receptor at a dose of 2.5 μg / mouse, and Het-R2 was less toxic than other types of prodrugs.

[0241] In summary, in the murine MC38 model, a prodrug of IL12-Fc conjugated to the IL12 receptor maintains the antitumor efficacy and enhances the safety of IL12-Fc. A low dose (2.5 μg) of Het IL12 prodrug conjugated to the IL12 receptor inhibited 130-150 mmHg of tumor cells. 3 The MC38 tumor with a tumor volume of 10 ...

[0242] Similar human versions of the various IL15 fusion proteins and prodrugs disclosed herein were also produced and tested in vitro. Production of the human proteins followed the same cloning, transfection, and purification protocols described above.

[0243] The results of SDS-PAGE electrophoresis of purified human fusion proteins incubated with or without MMP14 for 24 hours at 37° C. are shown in FIG.

[0244] The function of human Het-R1 and Het-R1 / R2 was measured using the HEK-Blue™ IL12 reporter cell line assay (Invivogen). HEK-Blue™ IL-12 cells were designed to detect bioactive human IL-12 by expressing a STAT4-inducible SEAP reporter gene. IL-12 binding to IL-12R on the surface of HEK-Blue™ IL-12 cells triggers a signaling cascade leading to the activation of STAT-4 with subsequent production of SEAP. QUANTI-Blue™ was used to measure HEK-Blue™ IL-12 cell activation and detect SEAP in cell supernatants.

[0245] The following HEK-Blue™ IL-12 cell reporter cell line assay was used: (1) Gently rinse HEK-Blue™ IL-12 cells in PBS and incubate in pre-warmed fresh test medium (DMEM, 4.5 g / l glucose, 2 mM L-glutamine, 10% (v / v) heat-inactivated PBS (30 min, 56 °C, ~1 x 10 6 The cells were suspended in 0.1 ml of 0.1 mM NaCl. (2) Samples were serially diluted in flat-bottom 96-well plates and incubated for 20-24 h at 37°C in a CO2 incubator with 50 μl of cell suspension (~50,000 cells) per well. (3) Incubate 20 μL of induced HEK-Blue™ IL-12 cell supernatant per well of a flat-bottom 96-well plate with 100 μL of remixed QUANTI-Blue™ solution for 15 min to 1 h in a 37°C incubator. (4) Determine the SEAP level using a spectrophotometer at 650 nm.

[0246] Results: Figure 14A shows that the IL12 activity of the prodrug of Het-R1 after MMP14 digestion is comparable to or greater than Het IL12. In contrast, Het-R1 without MMP14 digestion is less active, indicating that the prodrug blocking mechanism works as expected. Figure 14B shows that the activity of the Het-R1 / R2 prodrug after MMP14 digestion is comparable to Het IL12 and recombinant IL12. In contrast, Het-R1 / R2 without MMP14 digestion is less active, indicating that the prodrug blocking mechanism works as expected. <SEQリスト> SEQ ID No. 1: Mouse P40 subunit (without signal peptide) MWELEKDVYV VEVDWTPDAP GETVNLTCDT PEEDDITWTS DQRHGVIGSG KTLTITVKEF LDAGQYTCHK GGETLSHSHL LLHKKENGIW STEILKNFKN KTFLKCEAPN YSGRFTCSWL VQRNMDLKFN IKSSSSSPDS RAVTCGMASL SAEKVTLDQR DYEKYSVSCQ EDVTCPTAEE TLPIELALEA RQQNKYENYS TSFFIRDIIK PDPPKNLQMK PLKNSQVEVS WEYPDSWSTP HSYFSLKFFV RIQRKKEKMK ETEEGCNQKG AFLVEKTSTE VQCKGGNVCV QAQDRYYNSS CSKWACVPCR VRS SEQ ID No. 2: Human P40 subunit (without signal peptide) WELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATL SAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS SEQ ID No. 3: Mouse P35 subunit (without signal peptide) RVIPVSGPAR CLSQSRNLLK TTDDMVKTAR EKLKHYSCTA EDIDHEDITR DQTSTLKTCL PLELHKNESC LATRETSSTT RGSCLPPQKT SLMMTLCLGS IYEDLKMYQT EFQAINAALQ NNHHQQIILD KGMLVAIDEL MQSLNHNGET LRQKPPVGEA DPYRVKMKLC ILLHAFSTRV VTINRVMGYL SSA SEQ ID No. 4: Human P35 subunit (without signal peptide) RNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS SEQ ID No.5: Human Fc-hole DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No.6:ヒトFc knob DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No.7:Fc of IgG1 DKTHTCPPCP APELLGGPSV FLFPPKPKDQ LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPSRDELTK NQVSLTCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFLIGHT VDKSRWQQGN VFSCSVLHEA LHNHYTQKSL SLSPGK SEQ ID No.8:Rβ1 MDMMGLAGTS KHITFLLLCQ LGASGPGDGC CVEKTSFPEG ASGSPLGPRN LSCYRVSKTD YECSWQYDGP EDNVSHVLWC CFVPPNHTHT GQERCRYFSS GPDRTVQFWE QDGIPVLSKV NFWVESRLGN RTMKSQKISQ YLYNWTKTTP PLGHIKVSQS HRQLRMDWNV SEEAGAEVQF RRRMPTTNWT LGDCGPQVNS GSGVLGDIRG SMSESCLCPS ENMAQEIQIR RRRRLSSGAP GGPWSDWSMP VCVPPEVLP SEQ ID No. 9: βRβ1 MEPLVTWVVPLLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPPSSPWKLGDCGPQDDDTESCLCPLEMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPEN SEQ ID No. 10: mouseRβ2 MAQTVRECSL ALLFLFMWLL IKANIDVCKL GTVTVQPAPV IPLGSAANIS CSLNPKQGCS HYPSSNELIL LKFVNDVLVE NLHGKKVHDH TGHSSTFQVT NLSLGMTLFV CKLNCSNSQK KPPVPVCGVE ISVGVAPEPP QNISCVQEGE NGTVACSWNS GKVTYLKTNY TLQLSGPNNL TCQKQCFSDN RQNCNRLDLG INLSPDLAES RFIVRVTAIN DLGNSSSLPH TFTFLDIVIP LPPWDIRINF LNASGSRGTL QWEDEGQVVL NQLRYQPLNS TSWNMVNATN AKGKYDLRDL RPFTEYEFQI SSKLHLSGGS WSNWSESLRT RTPEEEP SEQ ID No. 11: βRβ2 MAHTFRGCSLAFMFIITWLLIKAKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQ LSGPKNLTWQKQCKDIYCDYLDFGINLTESPESNFTAKVTAVNSLGSSSSLPSTFTFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRYRPSNSRLWNMVNVTKAKGRHDLLDLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEP SEQ ID No. 12: Linker segment L1 GGGGSGGGGSGGGGS SEQ ID No. 13: Linker segment L2 (MMP14) GGGGSSGARYRWLTAGGGGS SEQ ID No. 14: Linker segment L2 GGGGSSGRSENIRTAGGGGS SEQ ID No. 15: Linker segment L2 (MMP14) GGGGSSGRAMHMYTAGGGGS SEQ ID No. 16: Linker segment L2 (MMP14) GGGGSSGAAMHMYTAGGGGS SEQ ID No. 17: Linker segment L2 (MMP14) GGGGSSGAIGFLRTAGGGGS SEQ ID No. 18: Linker segment L2 (MMP14) GGGGSSGASENIRTAGGGGS SEQ ID No. 19: Linker segment L2 (MMP14) GGGGSSGRPENIRTAGGGGS SEQ ID No. 20: Linker segment L2 (MMP14) GGGGSSGAPENIRTAGGGGS SEQ ID No. 21: Linker segment L2 (MMP14) GGGGSSGLISHSITAGGGGS SEQ ID No. 22: Linker segment L2 (MMP14) GGGGSSGNLRSKLTAGGGGS SEQ ID No. 23: Linker segment L2 (MMP14) GGGGSSGVFSIPLTAGGGGS SEQ ID No. 24: Linker segment L2 (MMP14) GGGGSSGIKYHSLTAGGGGS SEQ ID No. 25: Linker segment L2 (MMP14) SEQ ID No. 26: Linker segment L2 (MMP14) GGGGSSGRIGFLRTAGGGGS SEQ ID No. 27: Mouse P35 signal peptide SP1 MCQSRYLLFL ATLALLNHLS LA SEQ ID No. 28: Human P35 signal peptide SP1 MCPARSLLLVATLVLLDHLSLA SEQ ID No. 29: Mouse P40 signal peptide SP2 MCPQKLTISW FAIVLLVSPL MA SEQ ID No. 30: Human P40 signal peptide SP2 MCHQQLVISWFSLVFLASPLVAI SEQ ID No. 31: Mouse Homo IL-12 MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLHLKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPPKNLQ MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRSGGGGS GGGGSGGGGS RVIPVSGPAR CLSQSRNLLK TTDDMVKTAR EKLKHYSCTA EDIDHEDITR DQTSTLKTCL PLELHKNESC LATRETSST RGSCLPPQKT SLMMTLCLGS IYEDLKMYQT EFQAINAALQ NHNHQQIILD KGMLVAIDEL MQSLNHNGET LRQKPPVGEA DPYRVKMKLC ILLHAFSTRV VTINRVMGYL SSAGGGGSGG GGSGGGGSDK THTCPPCPAP ELLGGPSVFL FPPKPKDQLM ISRTPEVTCV VVVSHEDPE VKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYK TTPPVLDSDG SFLYSKLTVD KSRWQQGNVF SCSVLHEALH NHYTQKSLSL SPGK SEQ ID No.32:Homo IL12 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSGNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEI DHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDQ LMISRTPEVTVCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLYSKLTVDKSRWQQGNVFSCSVLHEALHNHYTQKSLSLPGK SEQ ID No.33: mouseHet IL-12 subunit1 MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLLHKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPPKNLQ MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No. 34: The IL12subunit1 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREK KDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.35: mouseHet IL12 subunit2 MCQSRYLLFL ATLALLNHLS LARVIPVSGP ARCLSQSRNL LKTTDDMVKT AREKLKHYSC TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS TTRGSCLPPQ KTSLMMTLCL GSIYEDLKMY QTEFQAINAA LQNHNHQQII LDKGMLVAID ELMQSLNHNG ETLRQKPPVG EADPYRVKMK LCILLHAFST RVVTINRVMG YLSSAGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVNHAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No.36: Het IL12 subunit2 MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 37: mouse Homo-R1 MDMMGLAGTS KHITFLLLCQ LGASGPGDGC CVEKTSFPEG ASGSPLGPRN LSCYRVSKTD YECSWQYDGP EDNVSHVLWC CFVPPNHTHT GQERCRYFSS GPDRTVQFWE QDGIPVLSKV NFWVESRLGN RTMKSQKISQ YLYNWTKTTP PLGHIKVSQS HRQLRMDWNV SEEAGAEVQF RRRMPTTNWT LGDCGPQVNS GSGVLGDIRG SMSESCLCPS ENMAQEIQIR RRRRLSSGAP GGPWSDWSMP VCVPPEVLPG GGGSSGRSEN IRTAGGGGSM WELEKDVYVV EVDWTPDAPG ETVNLTCDTP EEDDITWTSD QRHGVIGSGK TLTITVKEFL DAGQYTCHKG GETLSHSHLL LHKKENGIWS TEILKNFKNK TFLKCEAPNY SGRFTCSWLV QRNMDLKFNI KSSSSPDSR AVTCGMASLS AEKVTLDQRD YEKYSVSCQE DVTCPTAEET LPIELALEAR QQNKYENYST SFFIRDIIKP DPPKNLQMKP LKNSQVEVSW EYPDSWSTPH SYFSLKFFVR IQRKKEKMKE TEEGCNQKGA FLVEKTSTEV QCKGGNVCVQ AQDRYYNSSC SKWACVPCRV RSGGGGSGGG GSGGGGSRVI PVSGPARCLS QSRNLLKTTD DMVKTAREKL KHYSCTAEDI DHEDITRDQT STLKTCLPLE LHKNESCLAT RETSSTTRGS CLPPQKTSLM MTLCLGSIYE DLKMYQTEFQ AINAALQNHN HQQIILDKGM LVAIDELMQS LNHNGETLRQ KPPVGEADPY RVKMKLCILL HAFSTRVVTI NRVMGYLSSA GGGGSGGGGS GGGGSDKTHT CPPCPAPELL GGPSVFLFPP KPKDQLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NIGHTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DARTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFL YSKLTVDKSR WQQGNVFSCS VLHEALHNHY TQKSLSSLSPG K SEQ ID No.38:ヒトHomo-R1 SEQ ID No. 39: mouse Homo-R2 MAQTVRECSL ALLFLFMWLL IKANIDVCKL GTVTVQPAPV IPLGSAANIS CSLNPKQGCS HYPSSNELIL LKFVNDVLVE NLHGKKVHDH TGHSSTFQVT NLSLGMTLFV CKLNCSNSQK KPPVPVCGVE ISVGVAPEPP QNISCVQEGE NGTVACSWNS GKVTYLKTNY TLQLSGPNNL TCQKQCFSDN RQNCNRLDLG INLSPDLAES RFIVRVTAIN DLGNSSSLPH TFTFLDIVIP LPPWDIRINF LNASGSRGTL QWEDEGQVVL NQLRYQPLNS TSWNMVNATN AKGKYDLRDL RPFTEYEFQI SSKLHLSGGS WSNWSESLRT RTPEEEPGGG GSSGRSENIR TAGGGGSMWE LEKDVYVVEV DWTPDAPGET VNLTCDTPEE DDITWTSDQR HGVIGSGKTL TITVKEFLDA GQYTCHKGGE TLSHSHLLLH KKENGIWSTE ILKNFKNKTF LKCEAPNYSG RFTCSWLVQR NMDLKFNIKS SSSSPDSRAV TCGMASLSAE KVTLDQRDYE KYSVSCQEDV TCPTAEETLP IELALEARQQ NKYENYSTSF FIRDIIKPDP PKNLQMKPLK NSQVEVSWEY PDSWSTPHSY FSLKFFVRIQ RKKEKMKETE EGCNQKGAFL VEKTSTEVQC KGGNVCVQAQ DRYYNSSCSK WACVPCRVRS GGGGSGGGGS GGGGSRVIPV SGPARCLSQS RNLLKTTDDM VKTAREKLKH YSCTAEDIDH EDITRDQTST LKTCLPLELH KNEESCLATRE TSSTTRGSCL PPQKTSLMMT LCLGSIYEDL KMYQTEFQAI NAALQNHNHQ QIILDKGMLV AIDELMQSLN HNGETLRQKP PVGEADPYRV KMKLCILLHA FSTRVVTINR VMGYLSSAGG GGSGGGGSGG GGSDKTHTCP PCPAPELLGG PSVFLFPPKP KDQLMISRTP EVTCVVVDVS HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGFLYS KLTVDKSRWQ QGNVFSCSVL HEALHNHYTQ KSLSLSPGK SEQ ID No.40:ヒトHomo-R2 SEQ ID No.41:マウスHet-R1 / R2サブユニット1 MDMMGLAGTS KHITFLLLCQ LGASGPGDGC CVEKTSFPEG ASGSPLGPRN LSCYRVSKTD YECSWQYDGP EDNVSHVLWC CFVPPNHTHT GQERCRYFSS GPDRTVQFWE QDGIPVLSKV NFWVESRLGN RTMKSQKISQ YLYNWTKTTP PLGHIKVSQS HRQLRMDWNV SEEAGAEVQF RRRMPTTNWT LGDCGPQVNS GSGVLGDIRG SMSESCLCPS ENMAQEIQIR RRRRLSSGAP GGPWSDWSMP VCVPPEVLPG GGGSSGRSEN IRTAGGGGSM WELEKDVYVV EVDWTPDAPG ETVNLTCDTP EEDDITWTSD QRHGVIGSGK TLTITVKEFL DAGQYTCHKG GETLSHSHLL LHKKENGIWS TEILKNFKNK TFLKCEAPNY SGRFTCSWLV QRNMDLKFNI KSSSSSPDSR AVTCGMASLS AEKVTLDQRD YEKYSVSCQE DVTCPTAEET LPIELALEAR QQNKYENYST SFFIRDIIKP DPPKNLQMKP LKNSQVEVSW EYPDSWSTPH SYFSLKFFVR IQRKKEKMKE TEEGCNQKGA FLVEKTSTEV QCKGGNVCVQ AQDRYYNSSC SKWACVPCRV RSGGGGSGGG GSGGGGSDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP CRDELTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK SEQ ID No. 42: Human Het-R1 / R2 subunit 1 MEPLVTWVVPLLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRRHTPSPWKLGDCGPQDDDTESCLCPL EMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENGGGGSSGRSENIRTAGGGGSWELKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.43: μαυσHet-R1 / R2 subunit2 MAQTVRECSL ALLFLFMWLL IKANIDVCKL GTVTVQPAPV IPLGSAANIS CSLNPKQGCS HYPSSNELIL LKFVNDVLVE NLHGKKVHDH TGHSSTFQVT NLSLGMTLFV CKLNCSNSQK KPPVPVCGVE ISVGVAPEPP QNISCVQEGE NGTVACSWNS GKVTYLKTNY TLQLSGPNNL TCQKQCFSDN RQNCNRLDLG INLSPDLAES RFIVRVTAIN DLGNSSSLPH TFTFLDIVIP LPPWDIRINF LNASGSRGTL QWEDEGQVVL NQLRYQPLNS TSWNMVNATN AKGKYDLRDL RPFTEYEFQI SSKLHLSGGS WSNWSESLRT RTPEEEPGGG GSSGRSENIR TAGGGGSRVI PVSGPARCLS QSRNLLKTTD DMVKTAREKL KHYSCTAEDI DHEDITRDQT STLKTCLPLE LHKNESCLAT RETSSTTRGS CLPPQKTSLM MTLCLGSIYE DLKMYQTEFQ AINAALQNHN HQQIILDKGM LVAIDELMQS LNHNGETLRQ KPPVGEADPY RVKMKLCILL HAFSTRVVTI NRVMGYLSSA GGGGSGGGGS GGGGSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK SEQ ID No. 44: ヤHet-R1 / R2 subunit2 mahtfrgcslafmfiitwllikaKIDACKRGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESN FTAKVTAVNSLGSSSSLPSTFFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRRYRPSNSRLWNMVNVTKAKGRHDLDLLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPGGGGSSGRSENIRTAGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.45: mouseHet-R1 subunit1 MDMMGLAGTS KHITFLLLCQ LGASGPGDGC CVEKTSFPEG ASGSPLGPRN LSCYRVSKTD YECSWQYDGP EDNVSHVLWC CFVPPNHTHT GQERCRYFSS GPDRTVQFWE QDGIPVLSKV NFWVESRLGN RTMKSQKISQ YLYNWTKTTP PLGHIKVSQS HRQLRMDWNV SEEAGAEVQF RRRMPTTNWT LGDCGPQVNS GSGVLGDIRG SMSESCLCPS ENMAQEIQIR RRRRLSSGAP GGPWSDWSMP VCVPPEVLPG GGGSSGRSEN IRTAGGGGSM WELEKDVYVV EVDWTPDAPG ETVNLTCDTP EEDDITWTSD QRHGVIGSGK TLTITVKEFL DAGQYTCHKG GETLSHSHLL LHKKENGIWS TEILKNFKNK TFLKCEAPNY SGRFTCSWLV QRNMDLKFNI KSSSSSPDSR AVTCGMASLS AEKVTLDQRD YEKYSVSCQE DVTCPTAEET LPIELALEAR QQNKYENYST SFFIRDIIKP DPPKNLQMKP LKNSQVEVSW EYPDSWSTPH SYFSLKFFVR IQRKKEKMKE TEEGCNQKGA FLVEKTSTEV QCKGGNVCVQ AQDRYYNSSC SKWACVPCRV RSGGGGSGGG GSGGGGSDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP CRDELTKNQV SLWCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK SEQ ID No. 46: Human Het-R1 subunit 1 MEPLVTWVVPLLFLFLLSRQGAACRTSECCFQDPPYPDADSGSASGPRDLRCYRISSDRYECSWQYEGPTAGVSHFLRCCLSSGRCCYFAAGSATRLQFSDQAGVSVLYTVTLWVESWARNQTEKSPEVTLQLYNSVKYEPPLGDIKVSKLAGQLRMEWETPDNQVGAEVQFRHRTPSSPWKLGDCGPQDDDTESCLCPL EMNVAQEFQLRRRRLGSQGSSWSKWSSPVCVPPENGGGGSSGRSENIRTAGGGGSWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVT CGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPK PKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 47: Mouse Het-R1 subunit 2 MCQSRYLLFL ATLALLNHLS LARVIPVSGP ARCLSQSRNL LKTTDDMVKT AREKLKHYSC TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS TTRGSCLPPQ KTSLMMTLCL GSIYEDLKMY QTEFQAINAA LQNHNHQQII LDKGMLVAID ELMQSLNHNG ETLRQKPPVG EADPYRVKMK LCILLHAFST RVVTINRVMG YLSSAGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVHNAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVCT LPPSRDELTK NQVSLSCAVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLVSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No. 48: Human Het-R1 subunit 2 MCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDHEDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.49: mouseHet-R2 subunit1 MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD APGETVNLTC DTPEEDDITW TSDQRHGVIG SGKTLTITVK EFLDAGQYTC HKGGETLSHS HLLHLKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPPKNLQ MKPLKNSQVE VSWEYPDSWS TPHSYFSLKF FVRIQRKKEK MKETEEGCNQ KGAFLVEKTS TEVQCKGGNV CVQAQDRYYN SSCSKWACVP CRVRSGGGGS GGGGSGGGGS DKTHTCPPCP APELLGGPSV FLFPPKPKDT LMISRTPEVT CVVVDVSHED PEVKFNWYVD GVEVNHAKTK PREEQYNSTY RVVSVLTVLH QDWLNGKEYK CKVSNKALPA PIEKTISKAK GQPREPQVYT LPPCRDELTK NQVSLWCLVK GFYPSDIAVE WESNGQPENN YKTTPPVLDS DGSFFLYSKL TVDKSRWQQG NVFSCSVMHE ALHNHYTQKS LSLSPGK SEQ ID No.50: ヤHet-R2subunit1 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREK KDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No.51: mouseHet-R2 subunit2 MAQTVRECSL ALLFLFMWLL IKANIDVCKL GTVTVQPAPV IPLGSAANIS CSLNPKQGCS HYPSSNELIL LKFVNDVLVE NLHGKKVHDH TGHSSTFQVT NLSLGMTLFV CKLNCSNSQK KPPVPVCGVE ISVGVAPEPP QNISCVQEGE NGTVACSWNS GKVTYLKTNY TLQLSGPNNL TCQKQCFSDN RQNCNRLDLG INLSPDLAES RFIVRVTAIN DLGNSSSLPH TFTFLDIVIP LPPWDIRINF LNASGSRGTL QWEDEGQVVL NQLRYQPLNS TSWNMVNATN AKGKYDLRDL RPFTEYEFQI SSKLHLSGGS WSNWSESLRT RTPEEEPGGG GSSGRSENIR TAGGGGSRVI PVSGPARCLS QSRNLLKTTD DMVKTAREKL KHYSCTAEDI DHEDITRDQT STLKTCLPLE LHKNESCLAT RETSSTTRGS CLPPQKTSLM MTLCLGSIYE DLKMYQTEFQ AINAALQNHN HQQIILDKGM LVAIDELMQS LNHNGETLRQ KPPVGEADPY RVKMKLCILL HAFSTRVVTI NRVMGYLSSA GGGGSGGGGS GGGGSDKTHT CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE PQVCTLPPSR DELTKNQVSL SCAVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF LVSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK SEQ ID No.52: ヤHet-R2 subunit2 MAHTFRGCSLAFMFIITWLLIKADIACKRGGDVTVKPSHVILLGSTVNITCSLKPRQGCFHYSRRNKLILYKFDRRINFHHGHSLNSQVTGLPLGTTLFVCKLACINSDEIQICGAEIFVGVAPEQPQNLSCIQKGEQGTVACTWERGRDTHLYTEYTLQLSGPKNLTWQKQCKDIYCDYLDFGINLTPESPESN FTAKVTAVNSLGSSSSLPSTFFLDIVRPLPPWDIRIKFQKASVSRCTLYWRDEGLVLLNRLRRYRPSNSRLWNMVNVTKAKGRHDLDLLKPFTEYEFQISSKLHLYKGSWSDWSESLRAQTPEEEPGGGGSSGRSENIRTAGGGGSRNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKARQTLEFYPCTSEEIDH EDITKDKTSTVEACLPLELTKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNASGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0247] Applicant's disclosure is described herein in a preferred embodiment with reference to the Figures, in which like numerals represent the same or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," or similar terminology means that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar terminology throughout this specification may, but do not necessarily, all refer to the same embodiment.

[0248] The described functions, structures, or features of the disclosure of the present application may be combined in any suitable manner in one or more embodiments. In the description herein, numerous specific details are set forth to provide a thorough understanding of the embodiments of the present invention. However, one of ordinary skill in the art will recognize that the applicant's compositions and / or methods may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations have not been shown or described in detail to avoid obscuring aspects of the disclosure.

[0249] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.Any methods and materials similar and equivalent to those described herein can also be used in the practice or testing of this disclosure, but preferred methods and materials are described herein.The methods detailed herein may be carried out in any order that is logically possible, in addition to the specific order disclosed.

[0250] INCORPORATION BY REFERENCE References or citations to other documents, such as patents, patent applications, patent publications, journals, books, articles, web content, etc., are made in this disclosure. All such documents are incorporated herein by reference in their entirety for all purposes. Any material or portion thereof that is said to be incorporated herein by reference but that conflicts with existing definitions, statements, or other disclosed material expressly set forth herein, is incorporated only to the extent that no conflict occurs between the incorporated material and the material of this disclosure. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure.

[0251] equivalent The representative examples are intended to help illustrate the invention and are not intended, and should not be construed, as limiting the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of the specification, including the examples and references to the scientific and patent literature contained herein. The examples contain important additional information, exemplification, and guidance that can be adapted to the practice of this invention in its various embodiments, and equivalents thereof.

Claims

1. A fusion protein comprising, in order: M is an IL12 mask moiety, wherein said IL12 mask moiety is an IL-12 binding domain of the interleukin 12 receptor (IL12R), a truncated IL12R extracellular domain thereof, IL12Rβ1, IL12Rβ2, or both; L2 is a second linker segment that covalently attaches M to the first structural unit A; A is a first structural unit, wherein said first structural unit is one or two subunits of interleukin 12 (IL12) selected from the P35 and P40 subunits, wherein the P35 and P40 subunits are derived from a mammal selected from the group consisting of human, monkey, mouse, dog, rat, cow, pig, and sheep, and have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 1-4; B is a second structural unit, wherein the second structural unit is an antibody Fc fragment, wherein the second structural unit is located at the C-terminus of the fusion protein and has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID No. 5-7; and L1 is a first linker segment that covalently bonds a first structural unit A and a second structural unit B or bonds two subunits of the first structural unit A; A fusion protein comprising:

2. The fusion protein of claim 1, wherein the antibody Fc fragment comprises a human Fc fragment, a human IgG1, or the human IgG1 is a human Fc-knob or a human Fc-hole.

3. The fusion protein of claim 1, wherein the P35 subunit has an amino acid sequence as set forth in SEQ ID No. 3 or 4, and the P40 subunit has an amino acid sequence as set forth in SEQ ID No. 1 or 2.

4. The fusion protein according to any one of claims 1 to 3, wherein the first linker segment (L1) has an amino acid sequence as set forth in SEQ ID No.

12.

5. A fusion protein described in any one of claims 1 to 4, wherein the second linker segment L2 is recognizable and cleavable by a proteolytic enzyme specifically expressed in the tumor microenvironment.

6. A fusion protein described in any one of claims 1 to 5, wherein the second linker segment L2 is recognizable and cleavable by a matrix metalloproteinase.

7. The fusion protein according to claim 1, wherein the second linker segment L2 comprises an amino acid sequence set forth in SEQ ID No. 13-26.

8. The homodimeric or heterodimeric protein according to any one of claims 1 to 7, wherein the homodimeric or heterodimeric protein comprises: Monomeric homodimer: IL12Rβ1, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1, and Fc of human IgG1, and having the amino acid sequence set forth in SEQ ID No. 37; Monomeric homodimer: IL12Rβ1, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1, and Fc of human IgG1, and having the amino acid sequence set forth in SEQ ID No. 38; Monomeric homodimer: IL12Rβ2, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1, and Fc of human IgG1, and having the amino acid sequence set forth in SEQ ID No. 39; Monomeric homodimer: IL12Rβ2, linker segment L2, P40 subunit, linker segment L1, P35 subunit, linker segment L1, and Fc of human IgG1, and having the amino acid sequence set forth in SEQ ID No. 40; a first monomeric heterodimer: a first monomeric heterodimer of IL12Rβ1, a linker segment L2, a P40 subunit, a linker segment L1, and a first human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 41; a second monomeric heterodimer: a second monomeric heterodimer of IL12Rβ2, a linker segment L2, a P35 subunit, a linker segment L1, and a second human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 43; and a heterodimer of A first monomeric heterodimer: a first monomeric heterodimer of IL12Rβ1, a linker segment L2, a P40 subunit, a linker segment L1, a first human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 42; and a second monomeric heterodimer: a second monomeric heterodimer of IL12Rβ2, a linker segment L2, a P35 subunit, a linker segment L1, and a second human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 44; and a heterodimer of A first monomeric heterodimer: a first monomeric heterodimer comprising IL12Rβ1, a linker segment L2, a P40 subunit, a linker segment L1, a first human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 45; and a second monomeric heterodimer: a second monomeric heterodimer comprising a P35 subunit, a linker segment L1, a second human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 47; and a heterodimer of A first monomeric heterodimer: a first monomeric heterodimer comprising IL12Rβ1, a linker segment L2, a P40 subunit, a linker segment L1, a first human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 46; and a second monomeric heterodimer: a second monomeric heterodimer comprising a P35 subunit, a linker segment L1, a second human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 48; and a heterodimer of A first monomeric heterodimer: a first monomeric heterodimer comprising a P40 subunit, a linker segment L1, and a first human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 49; and a second monomeric heterodimer: a second monomeric heterodimer comprising IL12Rβ2, a linker segment L2, a P35 subunit, a linker segment L1, a second human Fc fragment, and having the amino acid sequence set forth in SEQ ID No. 51; or A first monomeric heterodimer: a first monomeric heterodimer of a P40 subunit, a linker segment L1, and a first human Fc fragment, the first monomeric heterodimer having the amino acid sequence set forth in SEQ ID No. 50; A second monomeric heterodimer: a second monomeric heterodimer of an IL12Rβ2, a linker segment L2, a P35 subunit, a linker segment L1, and a second human Fc fragment, the second monomeric heterodimer having the amino acid sequence set forth in SEQ ID No. 52; A heterodimer of A homodimeric or heterodimeric protein,

9. The homodimeric or heterodimeric protein described in claim 8, wherein the linker segment L2 is cleavable by a proteolytic enzyme in a tumor microenvironment.

10. A purified protein of any one of claims 1-9.

11. A pharmaceutical composition comprising any one of the proteins of claims 1-10 and a pharma- ceutically acceptable excipient, carrier, or diluent.

12. Use of any one of the proteins of claims 1-10, or the pharmaceutical composition of claim 11, in the preparation of a medicament for treating or alleviating a disease or condition selected from hyperplasia, solid tumor, or hematopoietic malignancy.

13. The use of claim 12, wherein the disease or disorder is selected from head and neck cancer, endometrial cancer, colon cancer, ovarian cancer, breast cancer, melanoma, lung cancer, kidney cancer, liver cancer, anal cancer, sarcoma, lymphoma, leukemia, brain tumor, gastric cancer, testicular cancer, pancreatic cancer, and thyroid cancer.

14. A cell line comprising a polynucleotide encoding any one of the proteins of claims 1-10.

15. A method for producing a protein, comprising: Providing an expression vector encoding the protein of any one of claims 1 to 10; Introducing the expression vector into a host cell by transient transfection or a stable cell line, wherein the host cell is 293F or CHO. Cultivating a host cell in a medium under conditions sufficient to express the protein; and purifying the protein from the host cell or culture medium; A method comprising:

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