Long-acting interleukin-15 fusion protein, preparation method therefor and use thereof

Abstract

Provided is a pharmaceutical composition comprising a long-acting interleukin-15 (IL-15) fusion protein complex for use in treating cancer in a subject.SOLUTION: Provided are pharmaceutical compositions comprising an Fc monomer and an IL-15 receptor alpha sushi domain, wherein the carboxyl terminus of the IL-15 receptor alpha sushi domain is linked to the amino terminus of the Fc monomer, and wherein the IL-15 receptor alpha sushi domain non-covalently interacts with an IL-15 fragment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention belongs to the field of biotechnology, and in particular relates to long-acting interleukin-15 fusion proteins, methods for their preparation, nucleic acids, plasmids and host cells required for the construction of long-acting proteins, corresponding pharmaceutical compositions and their uses.

[0002] Cross-reference to related applications This application claims priority to Chinese Patent Application No. 2020116429173 (titled "Long-acting interleukin-15 fusion protein, its preparation method and its application") filed with the State Intellectual Property Administration of China on December 30, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0003] IL-15, a cytokine of approximately 12-14 kD discovered by Grabstein et al. in 1994, plays a role in the normal immune response of the body, such as promoting the proliferation of T cells, B cells, and NK cells. Both IL-15 and IL-2 belong to the IL-2 family, whose receptor members, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21, all contain the γc chain. IL-15 and IL-2 share the IL-2 / IL-15Rβ (CD122) and γc receptor (CD132), while the specific receptor for IL-15 is IL-15Rα (CD125).

[0004] IL-15 signaling can occur via a heterotrimeric complex of IL-15Rα, IL-15Rβ, and γc. Recently, a new mechanism of IL-15 action has emerged: IL-15 and IL-15Rα are coexpressed on APCs (monocytes and dendritic cells), and IL-15 bound to IL-15Rα is transpresented to neighboring NK cells and CD8 T cells that express only the IL-15Rβγc receptor. IL-15 transpresentation is now considered the primary mechanism of action for IL-15 in vivo, particularly in tumor immune surveillance. IL-15Rα contains a sushi domain, which can bind to IL-15 and is required for the biological function of bound IL-15. However, IL-15 and IL-15Rα are primarily expressed on the surface of dendritic cells and monocytes, and are not expressed in the free state. Therefore, activation of the IL-15 signaling pathway likely activates downstream signaling pathways after cell-cell contact. IL-15 and IL-2 have similar functions in activating T cells, but do not activate Treg cells. Therefore, IL-15 may have fewer side effects and serve as a substitute for IL-2 in future clinical use. Given the promise of IL-15 in the field of tumor immunotherapy, the NIH is conducting its first research into IL-15 in tumor treatment and is moving it into clinical studies.

[0005] However, natural IL-15 has a small molecular weight and a short in vivo half-life, which makes it difficult to control repeated administration, leads to poor compliance, and is prone to problems such as systemic immune side effects. Therefore, there is an urgent need in the art for approaches to improve the in vivo half-life of IL-15 and promote or enhance its in vivo biological activity.

[0006] Currently, there are three long-acting technologies for protein drugs: PEG (polyethylene glycol) modification, HSA (human serum albumin) fusion, and Fc (human antibody Fc region) fusion. Each of these three technologies has its own weaknesses. A common and significant drawback is that the molecular weight of fused or modified protein drugs increases significantly, often significantly reducing the yield and clinical efficacy of fusion protein drugs. In the field of long-acting proteins, the inventors have used synthetic biology to innovatively develop a novel monomeric Fc based on antibody IgG Fc (CN 109705211 B; 202011161007.3). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] CN 109705211 B Summary of the Invention [Problem to be solved by the invention]

[0008] The primary objective of this application is to construct a long-acting interleukin-15 fusion protein incorporating monomeric Fc (sFc), IL-15, and the IL-15 receptor alpha sushi domain. This fusion protein, which utilizes the transpresentation mechanism of IL-15, potentially has better developability, a longer in vivo half-life, and better in vivo antitumor activity. [Means for solving the problem]

[0009] To achieve the above objectives, the present invention provides the following technical solutions: In a first aspect, provided is a long-acting interleukin-15 fusion protein comprising an Fc monomer, an IL-15 receptor alpha sushi domain, and an IL-15 functional fragment, wherein one end of the IL-15 receptor alpha sushi domain is linked to the Fc monomer and the other end of the IL-15 receptor alpha sushi domain is linked to the IL-15 functional fragment.

[0010] In a preferred embodiment of the long-acting interleukin-15 fusion protein, the C-terminus of the IL-15 receptor alpha sushi domain is linked to an Fc monomer, and the IL-15 receptor alpha sushi domain is linked to an IL-15 functional fragment via a covalent bond; preferably, the Fc monomer is linked to the IL-15 receptor alpha sushi domain via a linker peptide, and more preferably, the linker peptide is GGGGS or (GGGGS)3.

[0011] In the present invention, a covalent bond is a type of chemical bond in which two or more atoms share outer electrons, and in an ideal state, the bond reaches an electron saturated state and forms a relatively stable chemical structure. The strong interaction formed between the electrons shared by adjacent atoms is called a covalent bond.

[0012] A linker peptide refers to a polypeptide chain containing flexible amino acid residues, such as Gly, Ser, Ala, or Thr. The polypeptide chain should be of a distance suitable for linking two molecules so that they are correctly aligned relative to each other and maintain the desired activity. Suitable lengths for this purpose include at least one amino acid residue and no more than 30 amino acid residues. Preferably, the linker is approximately 1 to 30 amino acids long, with preferred linker lengths being 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. Furthermore, the amino acid residues selected for inclusion in the linker peptide should not exhibit properties that significantly affect the activity of the two linked molecules. Therefore, the linker peptide generally does not exhibit charges that conflict with the two linked molecules, affect internal folding, or form bonds or other interactions with amino acid residues in one or more monomers that significantly interfere with the binding of the receptor monomer domains. Linker peptides containing flexible amino acid residues include glycine-serine polymers (such as (GS)n, (GSGGS)n, (GGGS)n, and (GGGS)n, where n is an integer of at least 1), glycine-alanine polymers, alanine-serine polymers, and other flexible linker peptides known in the art, such as the linking sequence of the Shaker potassium channel.

[0013] In the present invention, an Fc monomer refers to such an Fc polypeptide that has a molecular weight that is approximately half that of a wild-type Fc region, retains the FcRn-binding and Protein A / G-binding properties of an antibody Fc region, and can be efficiently expressed in prokaryotic cells.

[0014] In a preferred embodiment of the above long-acting interleukin-15 fusion protein, the sequence of the Fc monomer comprises the amino acid sequence set forth in SEQ ID NO:1.

[0015] X0 is any amino acid selected from L and S; X1 is any amino acid selected from C, G, S, L, N, D, F, I, V, Y, Q, K, E, M and T; X2 is any amino acid selected from L, Q, N, D, Y, R, C, G, S, F, T, I, V, A, K and M; X3 is any amino acid selected from P, N, T, I, S, M, Q, R, L, G, V, A, E, D, Y, F and H; X4 is any amino acid selected from K, N, S, I, M, E, Q, L, V, A, H, D, Y and F; and X5 is any amino acid selected from M and Y; Preferably, the sequence of the Fc monomer further comprises the amino acid sequence set forth in SEQ ID NO:2.

[0016] The sushi domain of IL-15 receptor α in the present invention means that the extracellular domain of IL-15 receptor α begins with a cysteine ​​residue (C1) encoded by the first exon 2 and ends with a cysteine ​​residue (C4) encoded by the fourth exon 2, and both the C1 and C4 residues are included in the sushi domain. The present invention also encompasses amino acid sequences of IL-15 receptor α sushi domains formed by substitution, deletion, or addition of one or more amino acid residues and having corresponding activity.

[0017] In a preferred embodiment of the long-acting interleukin-15 fusion protein, the sequence of the IL-15 receptor alpha sushi domain comprises the amino acid sequence set forth in SEQ ID NO: 3; preferably, the sequence of the IL-15 functional fragment comprises the amino acid sequence set forth in SEQ ID NO: 4.

[0018] SEQ ID NO: 1 (amino acid sequence of monomeric Fc) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ PREPQVYTX0PPSRDELTKNQVSLX1CX2VKGFYPSDIAVEWESNGQPENNYKTTX3PVLDSDGSFFLYSX4LTVDKSRWQGNVFSCSVX5HEALHNHYTQKSLSLSPGK.

[0019] SEQ ID NO: 2 (amino acid sequence of monomeric Fc) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTSPPSRDELTKNQVSLRCHVKGFYPSDIAVEWESNGQPENNYKTTKPVLDSDGSFFLYSDLTVDKSRWQQGNVSCSVYHEALHNHYTQKSLSLSPGK.

[0020] SEQ ID NO: 3 (amino acid sequence of IL-15RαSu) ITCPPPMSVEHADIWKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR.

[0021] SEQ ID NO:4 (amino acid sequence of IL-15 mutant) NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS.

[0022] The IL-15 functional fragment of the present invention refers to a cytokine of approximately 12 to 14 kD discovered by Grabstein et al. in 1994 (JG Giri et al., EMBO J. 1994 Jun 15; 13(12):2822-2830), which can function in the normal immune response of the body, for example, by promoting the proliferation of T cells, B cells, and NK cells. The present invention also encompasses amino acid sequences of IL-15 functional fragments that have corresponding activity and are formed by the substitution, deletion, or addition of one or more amino acid residues.

[0023] In a second aspect of the present invention, provided is a method for constructing the long-acting interleukin-15 fusion protein described above, comprising: (a) Linking the Fc monomer to the IL-15 receptor α sushi domain via a linker peptide segment; (b) obtaining a functional fragment of IL-15; and (c) Co-expression or separate expression of (a) and (b) followed by in vitro protein assembly.

[0024] Co-transfection expression refers to mixing a vector containing an insert of an Fc monomer and an IL-15 receptor alpha sushi domain linked via a linker peptide segment with a vector containing an IL-15 functional fragment in an appropriate ratio, transfecting cells (e.g., Expi293 cells), culturing, expressing, and purifying (e.g., one-step purification using protein G) the resulting IL-15 fusion protein.

[0025] The in vitro protein assembly method involves transfecting cells (e.g., Expi293 cells) separately at an appropriate ratio with an Fc monomer, an IL-15 receptor α sushi domain vector, and an IL-15 functional fragment vector, all linked via a linker peptide segment, and then culturing and inducing these cells. The supernatants of the induced cells are mixed at an appropriate ratio and purified (e.g., by one-step purification using protein G) to obtain highly purified target proteins.

[0026] In a third aspect of the present invention, there is provided a nucleic acid molecule encoding the long-acting interleukin-15 fusion protein described above.

[0027] In a fourth aspect of the present invention, there is provided a plasmid comprising the nucleic acid molecule described above, operably linked to regulatory sequences such as promoters and enhancers.

[0028] In a fifth aspect of the present invention, a host cell containing the above-described plasmid is provided. The host cell of the present invention can be any prokaryotic or eukaryotic cell, including, but not limited to, bacterial cells (such as Escherichia coli and Bacillus subtilis), insect cells (e.g., by using a baculovirus expression system), and yeast or mammalian cells (such as CHO or BHK cell lines). Other suitable host cells are known to those skilled in the art.

[0029] In a sixth aspect of the present invention, there is provided a pharmaceutical composition comprising an effective prophylactic or therapeutic dose of the long-acting interleukin-15 fusion protein, the nucleic acid molecule, or the plasmid, and a pharmaceutically acceptable carrier. The composition may be in the form of a lyophilized solution, an aqueous solution, a liposome, or a capsule. The concentration of the fusion protein, or its nucleic acid molecule or plasmid, of the present invention may vary from about 0.1% to 100% (by weight).

[0030] In a seventh aspect of the present invention, there is provided a detection kit comprising the long-acting interleukin-15 fusion protein, the nucleic acid molecule, or the plasmid; preferably, the detection kit is used for detecting pathogens and tumor cells. Pathogens include viruses, bacteria, fungi, and parasitic infections. Tumor cells include various benign tumor cells, malignant tumor cells (i.e., cancer cells), solid tumor cells, and hematogenous cancer cells.

[0031] A beneficial effect of the present invention is to provide methods for diagnosing, preventing or treating diseases, which comprise administering to a subject the IL-15 fusion proteins, nucleic acid molecules, plasmids and pharmaceutical compositions of the present invention.

[0032] The diseases described in this invention are autoimmune diseases, inflammatory diseases, neurodegenerative diseases, cancer, or pathogen infections.

[0033] Preferably, the IL-15 fusion protein of the present invention can be used to treat cancer.Cancer in the present invention includes, but is not limited to, lymphoma, germinoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenoma, melanoma, and leukemia or lymphoid malignancies.More specific examples of the above cancers include squamous cell carcinoma (e.g., squamous cell carcinoma), lung cancer, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, gastrointestinal cancer, pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatocellular carcinoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, Vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, testicular cancer, esophageal cancer, bile duct tumors, head cancer, neck cancer, bone marrow stromal tumors, osteoclast tumors, multiple myeloma, osteolytic bone cancer, central nervous system tumors, brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, retinal neuroblastoma), nasopharyngeal cancer, basal cell carcinoma, bile duct cancer, Kaposi's sarcoma, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma).

[0034] Preferably, the IL-15 fusion proteins of the present invention can be used to treat pathogen infections, including, but not limited to, bacteria, fungi, viruses, and parasites.

[0035] Specific conditions that can be treated by the IL-15 fusion proteins of the present invention include, but are not limited to, congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other myocardial diseases, systemic lupus erythematosus, diabetes, spondylosis, synovial fibroblastic hyperplasia, bone loss, Paget's disease, disuse osteopenia, malnutrition, periodontal disease, familial splenic anemia, Langerhans cell histiocytosis, and Spinal cord injury, acute septic arthritis, osteomalacia, hypercortisolism, monostotic fibrous dysplasia, multiple fibrous dysplasia, periodontal reconstruction and fractures, sarcoidosis, bone metastasis / osteomalacia treatment and malignant hypercalcemia of body fluids, ankylosing spondylitis and other spondyloarthropathy, transplant rejection, viral infections, hematologic malignancies, Hodgkin's lymphoma, non-Hodgkin's lymphoma (Burkitt's lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia, lymphoplasmacytic leukemia), Lymphoid precursor neoplasms, B-cell acute lymphoblastic leukemia / lymphoma, T-cell acute lymphoblastic leukemia / lymphoma, thymoma, mature T-cell and NK cell neoplasms, peripheral T-cell leukemia, mature T-cell leukemia / T-cell lymphoma, large granular lymphocytic leukemia, Langerhans cell histiocytosis, myeloma with acute myeloid leukemia, mature acute myeloid leukemia (AML), differentiated acute myeloid leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, myelodysplastic syndrome, chronic myeloproliferative disorders, chronic myeloid leukemia, osteoporosis, hepatitis, HIV, AIDS, spondyloarthritis, Rheumatoid arthritis, inflammatory bowel disease (IBD), sepsis and septic shock, cicatricial enterocolitis, psoriasis, scleroderma, graft-versus-host disease (GVHD), allogeneic islet graft rejection, hematological malignancies such as multiple myeloma (MM), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML), tumor-associated inflammation, peripheral nerve injury, or demyelinating diseases.

[0036] The IL-15 fusion proteins, nucleic acid molecules, plasmids, and pharmaceutical compositions of the present invention can be administered to human or animal subjects via a variety of routes of administration, generally depending on the characteristics of the disease to be treated. Generally, any medically acceptable mode of administration can be used to practice the methods of the present invention, including oral, rectal, topical, intraocular, intrathoracic, intraventricular, intratracheal, nasal, transdermal, subcutaneous, intrathecal, intramuscular, intraperitoneal, intraperitoneal, intracranial, or intravenous infusion. [Brief explanation of the drawings]

[0037] [Figure 1] Figure 1 is a structural model diagram of the novel long-acting interleukin-15 fusion protein; [Figure 2] Figure 2 shows the reducing SDS-PAGE analysis and Coomassie brilliant blue staining of the novel long-acting interleukin-15 fusion protein; [Figure 3] Figure 3 shows high-performance liquid chromatography (HPLC) analysis of novel long-acting interleukin-15 fusion proteins, with analytes being FL115-2a (C) and FL115-2b (D) and control proteins being sFc (A) and Fc (b); [Figure 4] Figure 4 shows the biological activity of the novel long-acting interleukin-15 fusion protein evaluated by CTLL-2 proliferation assay, with the positive control being a fusion protein of IL-15 and IL-15 receptor α (positive control); [Figure 5] Figure 5 shows the in vivo antitumor effect of the novel long-acting interleukin-15 fusion protein FL115-2a in mice; [Figure 6] Figure 6 shows the effect of the novel long-acting interleukin-15 fusion protein of the present invention on the body weight of mice inoculated subcutaneously on the back with B16F10 melanoma tumors; [Figure 7]Figure 7 shows the effect of the novel long-acting interleukin-15 fusion protein of the present invention on the survival rate of mice inoculated subcutaneously on the back with B16F10 melanoma tumors; [Figure 8] Figure 8 shows the antitumor effect of a novel long-acting interleukin-15 fusion protein in mice and its effect on the body weight of mice subcutaneously inoculated with B16F10 melanoma tumors in the back. The positive controls were IL-15 Fc fusion protein (abbreviated as PC), IL15 KIH (IL-15 & IL15α Fc fusion protein), and WT-IL15, and the negative control was PBS; [Figure 9] Figure 9 shows the antitumor effect of the novel long-acting interleukin-15 fusion protein in vivo using five mice per group; [Figure 10] Figure 10 shows the antitumor effect of the novel long-acting interleukin-15 fusion protein on each group of mice. The tumor weight of each mouse was measured, and the statistical difference between the groups was analyzed by one-way ANOVA and statistical analysis. [Figure 11] Figure 11 shows that in mice administered the novel long-acting interleukin-15 fusion protein and then inoculated with B16F10 melanoma tumors, the number of Treg cells in vivo was reduced as analyzed by flow cytometry, as evidenced in particular by a decrease in Foxp3+CD25+; [Figure 12] Figure 12 shows flow cytometric analysis of the in vivo increase in NK cell numbers and increased secretion of IFN-γ and perforin in mice inoculated with B16F10 melanoma tumors after administration of a novel long-acting interleukin-15 fusion protein; [Figure 13] FIG. 13 shows flow cytometric analysis of in vivo IL6 increases in mice treated with a novel long-acting interleukin-15 fusion protein followed by inoculation with B16F10 melanoma tumors. DETAILED DESCRIPTION OF THE INVENTION

[0038] In order to allow those skilled in the art to better understand the solutions of the present invention, the technical solutions in the embodiments of the present invention are clearly and completely described below with examples. Obviously, the described embodiments are only a part, not all, of the embodiments of the present application. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without inventive efforts shall fall within the protection scope of the present application.

[0039] To better understand the present invention, the following definitions are provided: The above definitions are intended to include grammatical equivalents.

[0040] The term fusion protein has two distinct meanings. One is the recombinant expression product of two genes obtained through DNA recombinant technology, and the other is a group of proteins that mediate the fusion of two cell plasma membranes. For example, it is one of two glycoproteins contained in the outer leaflet of the lipid bilayer of Sendai virus, which mediates the fusion of the viral envelope with the host cell plasma membrane. The other glycoprotein is hemagglutinin-ceramidase. Two different proteins can be linked to form a polymer by chemical methods or gene fusion.

[0041] IL-15 is a soluble cytokine that acts as a chemokine on various immune cells and mediates inflammatory and immune responses in the body. Its biological function is similar to that of IL-2, which is produced by various cells.

[0042] As used herein, the term "monomer" refers to a molecule that can undergo polymerization to provide a structural unit of the basic structure of a polymer. The process by which multiple monomers combine to form a polymer is called polymerization.

[0043] As used herein, the terms "Fc" and "Fc monomer" encompass polypeptides containing antibody constant regions other than the first constant region of the immunoglobulin domain. Thus, Fc refers to the last two constant regions of the IgA, IgD, and IgG immunoglobulin domains, the last three constant regions of the IgE and IgM immunoglobulin domains, and the flexible hinge connecting the N-termini of these domains. For IgA and IgM, Fc may consist of the J chain. For IgG, Fc consists of immunoglobulin domains Cγ2 and Cγ3 and the hinge between Cγ1 and Cγ2. While the boundaries of the Fc region vary, the Fc region of the human IgG heavy chain is usually defined as including residues C226 or P230 at its carboxyl terminus, and its numbering follows the EU index of Kabat. Fc may refer to an isolated region or to a region in the context of an antibody, antibody fragment, or Fc fusion. Fc may refer to an antibody, an Fc fusion, or an Fc-containing protein or protein domain. Particularly preferred are Fc variants, including non-naturally occurring Fc variants obtained by synthetic biology.

[0044] A functional fragment is a type of compound that has a molecular structure intermediate between an amino acid and a protein and possesses strong biological activity in itself.

[0045] As used herein, the term "N-terminus," also known as the amino terminus, NH2 terminus, N-terminus, or amine terminus, refers to the initial end of a protein or polypeptide and the free amine group (-NH2) located at the end of the polypeptide. The term "C-terminus," also known as the carboxyl terminus, carboxyl group terminus, C-terminal tail, C-terminus, or COOH terminus, refers to the end of a protein or polypeptide and is terminated by a free carboxyl (-COOH) terminus.

[0046] As used herein, the term "amino acid" refers to one of the 20 naturally occurring amino acids or any non-natural analogue that may be located at a particular position. As used herein, the term "protein" refers to at least two covalently attached amino acids, including proteins, polypeptides, oligopeptides, and peptides. Proteins can be composed of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures called "analogs." Therefore, as used herein, the terms "amino acid" or "peptide residue" refer to both naturally occurring and synthetic amino acids. For example, for purposes of the present invention, homophenylalanine, citrulline, and norleucine are considered amino acids. The term "amino acid" also includes imino acid residues such as proline and hydroxyproline. Side chains can be in the (R) or (S) configuration. In preferred embodiments, amino acids are in the (S) or L-configuration. When non-naturally occurring side chains are used, non-amino acid substitutions may be used, for example, to prevent or retard in vivo degradation.

[0047] As used herein, the term "nucleic acid" refers to a polymer composed of nucleotide units (ribonucleotides, deoxynucleotides, related naturally occurring structural variants, and their synthetic non-naturally occurring analogs) linked together by phosphodiester bonds. Thus, the term includes, but is not limited to, nucleotide polymers in which the nucleotides and the linkages between them contain non-naturally occurring synthetic analogs, such as thiophosphate, aminophosphate, methylphosphate, chiral methylphosphate, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). For example, these polynucleotides can be synthesized using an automated DNA synthesizer. The term "oligonucleotide" generally refers to a short polynucleotide, usually less than about 50 nucleotides. When a nucleotide sequence is expressed as a DNA sequence (i.e., A, T, G, C), it should be understood that an RNA sequence (i.e., A, U, G, C) in which "T" is replaced with "U" is also included.

[0048] Conventional symbols are used to describe nucleotide sequences. The left end of a single-stranded nucleotide sequence is called the 5' end, and the left end of a double-stranded nucleotide sequence is called the 5' direction. The direction in which 5' to 3' nucleotides are added to the nascent RNA transcript is called the transcription direction. The DNA strand with the same base sequence as the mRNA is called the coding strand.

[0049] As used herein, the term "encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules by biological processes having either a defined nucleotide sequence or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA produced from that gene results in the protein in a cell or other biological system. For a gene or cDNA, the coding strand, whose nucleotide sequence is identical to the mRNA and is usually listed in a sequence listing, and the non-coding strand, used as a transcription template, can be said to encode the protein or other product of that gene or cDNA. Unless otherwise specified, the term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate and encode the same amino acid sequence. A nucleotide sequence encoding a protein or RNA may contain introns.

[0050] As used herein, the term "plasmid" refers to a plasmid artificially constructed based on a natural plasmid to be suitable for laboratory manipulation. A transformed host cell can be produced by introducing a nucleic acid molecule into a host cell. A vector can contain a nucleic acid sequence that enables it to replicate in a host cell, such as an origin of replication, and can also contain one or more selectable marker genes and other genetic elements known in the art.

[0051] As used herein, the term "host cell", also called recipient cell, refers to a host cell that receives foreign genes during transformation and transduction (infection).

[0052] As used herein, the term "pharmaceutically acceptable carrier" refers to conventional pharmaceutically acceptable carriers. Remington's Pharmaceutical Sciences, E.W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes compositions and preparations suitable for drug delivery of one or more therapeutic compounds or molecules (such as one or more antibodies) and additional agents.

[0053] As used herein, "diagnosis" of a disease refers to diagnosing a patient's condition and its onset after testing. "Prevention" of a disease refers to inhibiting the full onset of a disease. "Treatment" refers to therapeutic intervention to ameliorate signs or symptoms of a disease or pathological condition after it has begun to develop.

[0054] As used herein, the term "administration" refers to selecting an appropriate route for introducing a substance into a subject. For example, if the selected route is intravenous, the composition is administered by introducing the substance into the subject's vein.

[0055] As used herein, the term "effective prophylactic / therapeutic amount" refers to an amount of a particular agent sufficient to achieve the desired effect in a subject treated with the agent. The exact dosage will vary depending on the purpose of treatment and can be determined by one of ordinary skill in the art using well-known techniques. Dosage ranges from 0.01 to 100 mg / kg body weight or more, e.g., 0.1, 1, 10, or 50 mg / kg body weight, preferably 1 to 10 mg / kg. As is well known to those skilled in the art, adjustments may be necessary depending on factors such as antibody or Fc fusion degradation, systemic or local drug delivery, and the rate of new protease synthesis, as well as age, body weight, general health, sex, diet, administration time, drug interactions, and disease severity, and can be determined by one of ordinary skill in the art using routine experimentation. Such agents include the monomeric Fc domain molecules described herein. In one non-limiting example, this may be the amount of an HIV-specific monomeric Fc domain (or an HIV-specific CH3 domain molecule) to prevent, treat, or ameliorate HIV infection. Ideally, a therapeutically effective amount of an antibody is an amount sufficient to prevent, treat, or ameliorate infection or disease in a subject, such as that resulting from HIV infection, without causing significant cytotoxic effects in the subject. A therapeutically effective amount of an agent to prevent, ameliorate, and / or treat a subject will vary depending on the subject being treated, the type and severity of the indisposition, and the mode of administration of the therapeutic composition.

[0056] As used herein, the term "autoimmune disease" refers to a disease in which the immune system mounts an immune response (e.g., a B cell response or a T cell response) to a certain antigen (i.e., a self-antigen) in a normal host, resulting in subsequent tissue damage. The self-antigen may be derived from host cells or from commensal organisms, such as microorganisms that normally colonize mucosal surfaces (called commensals). Autoimmune diseases that affect mammals include rheumatoid arthritis, juvenile microarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjögren's syndrome, multiple sclerosis, experimental autoimmune encephalomyelitis, inflammatory bowel disease (e.g., Crohn's disease and ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes mellitus, myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture's syndrome, Addison's disease, systemic sclerosis, polymyositis, dermatomyositis, autoimmune hemolytic anemia, and pernicious anemia.

[0057] As used herein, the term "virus" refers to viruses from the following virus families, but is not limited to: Retroviridae (e.g., human immunodeficiency virus (HIV) and human T-cell leukemia virus (HTLV)); Picornaviridae (e.g., Picornaviridae (e.g., poliovirus, hepatitis A virus, hepatitis C virus, enterovirus, human coxsackievirus, rhinovirus, echovirus, foot-and-mouth disease virus, etc.); Kalmyxoviridae (e.g., viral strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus, etc.); Flaviviridae (e.g., dengue virus, yellow fever virus, dengue virus, yellow fever virus, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, other encephalitis viruses, etc.); Coronaviridae (e.g., coronavirus, severe acute respiratory syndrome (SARS) virus, etc.); Rhabdoviridae (e.g.,Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus (RSV)); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, Sin Nombre virus, Rift Valley fever virus, Bunyaviridae, phlebovirus, Nairovirus, etc.); Arenaviridae (e.g., hemorrhagic fever viruses, Machupo virus, Junin virus, etc.); Reoviridae (e.g., reovirus, orbivirus, rotavirus, etc.); Birnaviridae; Hepadnaviridae (e.g., hepatitis B virus, etc.); Parvoviridae (e.g., parvovirus); Papoviridae (e.g., papillomavirus, poliovirus, etc.) Adenoviridae (e.g., most adenoviruses, such as adeno-associated viruses); Herpesviridae (e.g., other herpesviruses, including herpes simplex viruses (HSV-1 and HSV-2), cytomegalovirus (CMV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), and HSV-6); Poxviridae (e.g., smallpox virus, vaccinia virus, and poxvirus); Iridoviridae (e.g., African swine fever virus); Viridae (e.g., Ebola virus and Marburg virus), Caliciviridae (e.g., Norwalk virus), and unclassified viruses (e.g., the causative agent of spongiform encephalopathy, the causative agent of hepatitis delta (thought to be a defective satellite of hepatitis B virus), and astrovirus).

[0058] As used herein, the term "bacteria" refers to Helicobacter pylori, Borrelia burgdorferi, Legionella pneumophila, mycobacteria (M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M.gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (group A streptococci), Streptococcus agalactiae (group B streptococci), Streptococcus (Viridans group streptococci), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic bacteria), Streptococcus pneumoniae pneumoniae, pathogenic Campylobacter, Enterococcus, Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium, classical swine fever virus, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides, Clostridium, Streptomyces maltophilia These include, but are not limited to, Treponema pallidum, Treponema pallidum, Leptospira, and Actinomyces israelli.

[0059] As used herein, the term "fungus" includes, but is not limited to, fungi from the group consisting of Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, or Candida albicans.

[0060] As used herein, "parasite" refers to, but is not limited to, Plasmodium or Toxoplasma gondii.

[0061] As used herein, "cancer" refers to a solid tumor or a hematogenous cancer. The solid tumor according to the present invention is a sarcoma or carcinoma, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma or other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, radioadenocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, nephroblastoma, cervical cancer, testicular tumor, bladder cancer, or a tumor of the central nervous system (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal gland, hemangioblastoma, acoustic neuroma, oligodendroglioma, hemangioma, melanoma, neuroblastoma, retinoblastoma, etc.). Hematogenous cancers according to the present invention are leukemias, such as acute leukemia (acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia, erythroblastic leukemia, etc.) and chronic leukemia (chronic myelocytic (granulocytic) leukemia, chronic granulocytic leukemia, chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, myelodysplasia.

[0062] Unless otherwise specified, 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 disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. It should be understood that all base or amino acid sizes and all molecular weight or molecular weight values ​​given for nucleic acids or polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and materials are described below. The term "comprises" means "includes." All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification (including explanations of terms) will control. Furthermore, the materials, methods, and examples are illustrative only and not limiting.

[0063] Standard recombinant DNA and molecular cloning techniques used in the examples are well known in the art (Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience), and materials and methods suitable for growing microorganisms are well known in the art. Major chemical and biochemical reagents were purchased from KAPA Biosystems, New England Biolabs, TransGen Biotech, Thermo Fisher Scientific, OMEGA bio-tek, etc.

[0064] The present invention will be described in detail below with reference to specific examples. Example 1. Design and preparation of a novel long-acting interleukin-15 fusion protein A schematic diagram of the designed novel long-acting interleukin-15 fusion proteins is shown in Figure 1. Genes encoding IL15Rα-GGGGS-sFc (SEQ ID NO: 5), IL15Rα-(GGGGS)3-sFc (SEQ ID NO: 6), IL15Rα-Fc (SEQ ID NO: 7), and IL15(N72D) (sequence SEQ ID NO: 4) were synthesized by Genescript, Nanjing. The genes were cloned into the eukaryotic expression vector PTT (purchased from Thermo Fisher Scientific). The successfully constructed plasmids were subjected to maxi-preparation and transiently co-transfected into Expi293 cells (purchased from Thermo Fisher) for expression (wherein IL15Rα-(GGGGS)3-sFc was co-transfected with IL15(N72D) to obtain FL115-2a), IL15Rα-GGGGS-sFc was co-transfected with IL15(N72D) to obtain FL115-2b, and IL15Rα-Fc was co-transfected with IL15(N72D) to obtain an Fc fusion protein of IL-15), and purified using protein G resin (purchased from GE Healthcare) (for specific transformation, expression, and purification methods, see YING, Tianlei et al., JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287(23):19399-19408). The purity of the purified protein was verified by SDS-PAGE gel, which showed that the protein was of good purity, there were no unnecessary bands, and the desired bands were correct (see Figure 2).

[0065] Example 2. Physicochemical properties of novel long-acting interleukin-15 fusion proteins Novel long-acting interleukin-15 fusion proteins (FL115-2a and FL115-2b) were expressed and purified according to the method described in Example 1. IL-15 Fc fusion proteins were also similarly designed and constructed, and expressed and purified using the same expression system and method to obtain the relevant proteins. Purification was performed using protein G resin (for specific transformation, expression, and purification methods, see YING, Tianlei et al., JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287(23):19399-19408). The yield of the novel long-acting interleukin-15 fusion proteins was up to 10 mg / L, which is significantly superior to the 1 mg / L yield of IL-15 Fc fusion proteins and theoretically offers significant advantages in terms of industrial production costs. Furthermore, to confirm the homogeneity of the above proteins, 100 μl of each aliquot was subjected to high-performance liquid chromatography (HPLC). As shown in Figure 3, the novel long-acting interleukin-15 fusion proteins were homogeneous in nature and existed in a monomeric form. FL115-2a and FL115-2b exhibited sFc and IL15Rα linked via GGGGS and (GGGGS)3 peptide segments, respectively. These results indicated that the novel long-acting interleukin-15 fusion proteins have better development potential.

[0066] Example 3. Evaluation of the biological activity of novel long-acting interleukin-15 fusion proteins in the CTLL-2 cell line To evaluate the biological activity of the novel long-acting interleukin-15 fusion protein, a proliferation assay of the CTLL-2 cell line was used (Santos-Savio A. et al., Biotechn Aplic. 2000, 17:221-4). Biological activity was measured by stimulating the proliferation of these cells using mitochondrial staining with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (purchased from Sigma) (see Mossman TJ Immunol. Methods. 1983, 65(1-2):55-63 for specific methods) according to the following procedure. Serial dilutions of FL115-2a, FL115-2b, positive controls (IL15 & IL15Rα), and sFc were prepared in 96-well culture plates (purchased from Costar, USA) starting at 4 μg / mL in 50 μL of RPMI medium (purchased from Gibco) supplemented with 10% fetal bovine serum (FBS) (purchased from Gibco) and 50 μg / mL gentamicin (purchased from Sigma). CTLL-2 cells (purchased from ATCC) were washed five times with RPMI medium and added to the plates at 5 × 10 cells / well in a volume of 50 μL. Incubation was performed at 37°C, 5% CO2, and 98% relative humidity for 72 hours. Cell viability was measured by MTT staining. sFc had no biological activity as it did not induce proliferation of CTLL-2 cells, which differed from FL115-2a, FL115-2b and the positive control (IL15 & IL15Rα), which stimulated proliferation in a dose-dependent manner (see Figure 4).

[0067] Example 4. In vivo pharmacodynamic validation of a novel long-acting interleukin-15 fusion protein To evaluate the pharmacodynamics of the novel long-acting interleukin-15 fusion protein, 6- to 8-week-old female C57BL / 6 mice (purchased from Jiangsu GemPharmatech Co., Ltd.) were injected with 5 x 10 B16F10 mouse melanoma cells in the right dorsal region. On days 1 and 8 after inoculation, mice were treated with various doses of FL115-2a-1, a positive control (IL15 & IL15Rα), wild-type IL15 (WT-IL15), or a negative control (PBS). The corresponding FL115-2a-2 was administered every other day after tumor inoculation. Tumor size was measured on the indicated days. Experimental results are the mean ± standard deviation of five mice per group. Statistical significance of the experimental data was analyzed by one-way analysis of variance, with ** indicating p<0.01. The positive control was a fusion protein of IL-15 and IL-15 receptor α (positive control), the WT-IL15 group and the PBS group were controls, FL115-2a-1 represented administration on days 1 and 8, and FL115-2a-2 represented administration every other day.

[0068] Experimental results showed that the administration mode of FL115-2a-1 and FL115-2a-2 did not affect the antitumor efficacy of the novel long-acting interleukin-15 fusion protein in vivo, and the two groups showed very clear antitumor effects with statistically significant differences compared to the negative control group. This may be due to the extended half-life of IL-15 in vivo, as shown in Figure 5. Furthermore, weight measurements of mice in each group confirmed the safety of the fusion protein, as shown in Figure 6. The FL115-2a-1 and FL115-2a-2 groups exhibited favorable survival rates, as shown in Figure 7.

[0069] Example 5. A novel long-acting interleukin-15 fusion protein exhibited efficient antitumor activity in vivo To evaluate the in vivo antitumor efficacy of the novel long-acting interleukin-15 fusion protein, we established a mouse tumor model by subcutaneously implanting B16F10 mouse melanoma cells into the dorsal skin of 6- to 8-week-old female C57BL / 6 mice (purchased from Jiangsu GemPharmatech Co., Ltd.) according to the experimental design described above for pharmacodynamic validation. After model establishment, FL115-2a, PC (IL-15 Fc fusion protein), IL15KIH (IL-15 & IL15Rα Fc fusion protein), WT-IL15, and negative control PBS were injected into the tail vein on days 1 and 8, and tumor size was measured every other day. As shown in Figure 8, FL115-2a demonstrated comparable in vivo tumor growth inhibition effects to PC and showed statistically significant differences from the negative control group. There was no effect on mouse body weight in any group, demonstrating the in vivo safety of the novel long-acting interleukin-15 fusion protein in mice, as shown in Figure 8. Furthermore, tumor suppression was analyzed for five mice in each group. The tumor suppression effect in the FL115-2a group was consistent, with a maximum suppression rate of 62% (Figure 9), demonstrating significant antitumor activity. The mice were dissected, and tumors were removed and weighed. FL115-2a demonstrated effective antitumor activity in vivo, demonstrating significant tumor suppression in the mice, with significant differences in both size and weight compared to the negative control group (Figure 10).

[0070] Example 6. Analysis of cell phenotype by flow cytometry After anesthesia, peripheral venous blood was collected from the orbital vein of each mouse and immediately anticoagulated and erythrocyte-lysed. After humanely sacrificing the mouse, the spleen was immediately removed. Spleen cells were collected and filtered through a 70 μM nylon sieve (Falcon). After erythrocyte lysis, a single-cell suspension of spleen was obtained. Furthermore, tumor tissue was removed, gently disrupted with tweezers, and then digested with 0.2 mg / ml collagenase IV (Sigma-Aldrich) and 0.1 mg / ml DNAse I (Sigma-Aldrich) at 37°C for 15 minutes. The single-cell suspension was collected, and the remaining tumor tissue was digested with the above enzyme solution for 25 minutes. The combined single-cell suspension was filtered through a 70-micron nylon mesh. Peripheral blood mononuclear cells, spleen cells, and tumor cells were stained with flow antibodies (BD), fixed in 4% paraformaldehyde solution (Sigma-Aldrich), and either stored in the dark or immediately loaded onto a flow cytometer (BD) to detect cell phenotypes. After antibody staining, flow cytometry analysis revealed a decrease in the number of Treg cells in tumor tissue from FL115-2a-treated mice, as evidenced by a decrease in Foxp3+CD25+ cells (Figure 11). Furthermore, the number of NK cells and the secretion of IFN-γ and perforin increased. These experimental results suggest that the novel long-acting interleukin-15 fusion protein not only reduces the number of Treg cells and their suppressive effect, but also significantly promotes the infiltration of NK cells into tumor tissue and significantly increases the secretion of IFN-γ and perforin. This further suggested that the novel long-acting interleukin-15 fusion protein could effectively activate the immune system to kill tumor cells and improve the tumor-suppressing effect of IL-15 in vivo, as shown in Figure 12. Furthermore, flow cytometry also demonstrated that the novel long-acting interleukin-15 fusion protein increased IL-6 expression, as shown in Figure 13.

[0071] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. It should be noted that the above description is only about the preferred embodiments of the present invention, and those skilled in the art may make some improvements and additions without departing from the principles of the present invention, and these improvements and additions should also be considered within the protection scope of the present invention. Many modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited by the embodiments shown herein, but is accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A long-acting interleukin-15 fusion protein comprising an Fc monomer, an IL-15 receptor alpha sushi domain, and an IL-15 functional fragment, wherein one end of the IL-15 receptor alpha sushi domain is linked to the Fc monomer and the other end of the IL-15 receptor alpha sushi domain is linked to the IL-15 functional fragment.

2. 2. The long-acting interleukin-15 fusion protein of claim 1, wherein the C-terminus of the IL-15 receptor alpha sushi domain is linked to an Fc monomer, and the IL-15 receptor alpha sushi domain is linked to an IL-15 functional fragment via a covalent bond.

3. The Fc monomer is linked to the IL-15 receptor alpha sushi domain via a linker peptide, where the linker peptide is GGGGS or (GGGGS) 3 3. The long-acting interleukin-15 fusion protein of claim 2, wherein

4. 4. The long-acting interleukin-15 fusion protein according to any one of claims 1 to 3, wherein the sequence of the Fc monomer comprises the amino acid sequence set forth in SEQ ID NO:

1. X0 is any amino acid selected from L and S; X1 is any amino acid selected from C, G, S, L, N, D, F, I, V, Y, Q, K, E, M and T; X2 is any amino acid selected from L, Q, N, D, Y, R, C, G, S, F, T, I, V, A, K and M; X3 is any amino acid selected from P, N, T, I, S, M, Q, R, L, G, V, A, E, D, Y, F and H; X4 is any amino acid selected from K, N, S, I, M, E, Q, L, V, A, H, D, Y and F; and X5 is any amino acid selected from M and Y.

5. 5. The long-acting interleukin-15 fusion protein of claim 4, wherein the sequence of the Fc monomer further comprises the amino acid sequence set forth in SEQ ID NO:

2.

6. 6. The long-acting interleukin-15 fusion protein of any one of claims 1 to 5, wherein the sequence of the IL-15 receptor alpha sushi domain comprises the amino acid sequence set forth in SEQ ID NO: 3; and the sequence of the IL-15 functional fragment comprises the amino acid sequence set forth in SEQ ID NO:

4.

7. 10. A method for constructing a long-acting interleukin-15 fusion protein according to any one of claims 1 to 6, comprising: (a) linking an Fc monomer to the IL-15 receptor alpha sushi domain via a linker peptide segment; (b) obtaining a functional fragment of IL-15; and (c) Co-expression or separate expression of (a) and (b) followed by in vitro protein assembly.

8. A nucleic acid molecule encoding any of the long-acting interleukin-15 fusion proteins of any of claims 1 to 7.

9. A plasmid comprising the nucleic acid molecule of claim 8.

10. A host cell comprising the plasmid of claim 9.

11. A pharmaceutical composition comprising a prophylactically or therapeutically effective amount of any of the long-acting interleukin-15 fusion proteins of claims 1 to 6, the nucleic acid molecule of claim 8, or the plasmid of claim 9, and a pharmaceutically acceptable carrier.

12. A detection kit comprising the long-acting interleukin-15 fusion protein of any one of claims 1 to 6, the nucleic acid molecule of claim 8, or the plasmid of claim 9.

13. 13. The detection kit according to claim 12, which is used for detecting pathogens and tumor cells.

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