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WO2015103928A1 - Il-15异源二聚体蛋白及其用途 - Google Patents

Il-15异源二聚体蛋白及其用途 Download PDF

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WO2015103928A1
WO2015103928A1 PCT/CN2014/094947 CN2014094947W WO2015103928A1 WO 2015103928 A1 WO2015103928 A1 WO 2015103928A1 CN 2014094947 W CN2014094947 W CN 2014094947W WO 2015103928 A1 WO2015103928 A1 WO 2015103928A1
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protein
seq
variant
group
sequence
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PCT/CN2014/094947
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English (en)
French (fr)
Inventor
屈向东
叶鑫
袁纪军
张蕾
陈侃
张连山
曹国庆
许志宾
陶维康
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上海恒瑞医药有限公司
江苏恒瑞医药股份有限公司
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First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=53523533&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=WO2015103928(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by 上海恒瑞医药有限公司, 江苏恒瑞医药股份有限公司 filed Critical 上海恒瑞医药有限公司
Priority to CA2935599A priority Critical patent/CA2935599A1/en
Priority to EP14878154.5A priority patent/EP3093295B1/en
Priority to CN201480010798.6A priority patent/CN105189562B/zh
Priority to KR1020167020066A priority patent/KR20160103058A/ko
Priority to AU2014377106A priority patent/AU2014377106B2/en
Priority to JP2016544083A priority patent/JP6484634B2/ja
Priority to US15/109,920 priority patent/US10206980B2/en
Priority to MX2016008631A priority patent/MX2016008631A/es
Priority to RU2016130447A priority patent/RU2689717C2/ru
Publication of WO2015103928A1 publication Critical patent/WO2015103928A1/zh

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    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
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Definitions

  • the present invention relates to an IL-15 heterodimeric protein and use thereof, and further to an IL-15/IL-15R ⁇ heterodimeric protein complex, and as a therapeutic agent, particularly as cancer and autoimmunity The use of disease therapeutics.
  • Interleukin-15 is a cytokine of about 12-14kD discovered by Grabstein et al. in 1994. It can play a role in the body's normal immune response, such as promoting T cells, B cells, and natural killing. (NK) cell proliferation.
  • IL-15 belongs to a member of the four small four-helix bundle family of cytokines. IL-15 needs to exert biological activity by binding to its receptor.
  • the IL-15 receptor consists of three receptor subunits: IL-15 receptor alpha (IL-15R alpha), IL-2 receptor beta (IL-2R beta, also known as IL-15R beta or CD122) and gamma c (also known as CD132).
  • IL-15R ⁇ contains a Sushi domain that binds to IL-15 and is required for the biological function of IL-15 after binding.
  • IL-15 and its receptor IL-15R ⁇ form a complex, which can significantly enhance the biological activity of IL-15.
  • the IL-15/IL-15R ⁇ complex stimulates memory CD8+ T cells to proliferate and maintain their survival more than 10 times stronger than IL-15 alone, and the mechanism may be related to cis-presentation.
  • IL-15 Due to the good expectations of IL-15 in the field of tumor immunotherapy, NIH first conducted research on IL-15 treatment of tumors and tried to push it into the clinical phase I study. However, IL-15 has a small molecular weight and a half-life phase in vivo, and the repeated administration dose is not well controlled, and it is easy to cause systemic immune side effects. Therefore, there is an urgent need for a way to increase the half-life of IL-15 in vivo, or to promote or enhance the biological activity in vivo.
  • IL-15 immunotherapy such as the IL-15-hIgG4Fc homodimeric protein involved in the CN100334112C (Shanghai Haixin Biotechnology Co., Ltd.) patent.
  • Treatment of microbial infections such as the IL-15-Fc fusion expression system and its use in the CN1942481A (Swiss Hoffman-Roche) patent; such as the IL-15Ra of the CN101360827B (French National Institute of Medicine and Health) patent (sushi+domain)-IL-15 fusion protein and its use in tumor therapy.
  • the heterodimeric molecules of the present application have better stability, extended in vivo half-life, and increased biological activity by increasing intramolecular interactions.
  • the use of targeted immunocytokines and targeted immunocytokines in the treatment of tumors and autoimmune diseases can be produced by fusion or insertion of functional polypeptides on the basis of the molecular design of the present application by methods well known in the art.
  • the invention designs and prepares a protein molecule having an in vivo half-life extension, an increased in vitro activity and a significant tumor suppressing activity by a genetic engineering method.
  • the present invention provides an IL-15 heterodimeric protein comprising:
  • protein (I) is recombined from IL-15 or a variant thereof and a first Fc variant
  • the protein ( ⁇ ) is a second Fc variant, or is recombined from IL-15R ⁇ or a variant thereof and a second Fc variant;
  • Protein (I) and protein ( ⁇ ) form a stable heterodimeric protein by interaction between the first Fc variant and the second Fc variant.
  • recombinant refers to the expression of recombinant protein after recombination by genetic engineering.
  • the first Fc variant and the second Fc variant are linked at the C-terminus of the proteins IL-15 and IL-15R ⁇ .
  • an IL-15 heterodimeric protein is provided, wherein the IL-15 sequence is SEQ ID NO: 1.
  • an IL-15 heterodimeric protein wherein said protein ( ⁇ ) is a second Fc variant.
  • an IL-15 heterodimeric protein wherein the protein ( ⁇ ) is recombinantly composed of IL-15R ⁇ or a variant thereof and a second Fc variant.
  • an IL-15 heterodimeric protein wherein the IL-15R ⁇ variant is an IL-15R ⁇ extracellular domain portion or a functional fragment thereof, and the functional fragment is preferably It is a shortened form of 65-120 amino acids of the IL-15R ⁇ extracellular domain, more preferably a shortened form of 65-102 amino acids.
  • an IL-15 heterodimeric protein wherein the sequence of the IL-15R ⁇ variant is selected from the group consisting of SEQ ID NOs: 2-7.
  • an IL-15 heterodimeric protein wherein the IL-15R ⁇ variant sequence is selected from the group consisting of SEQ ID NOs: 3-7.
  • an IL-15 heterodimeric protein wherein the first Fc variant and the second Fc variant are respectively selected from a Knob-modified Fc and a Hole-modified Fc, or The first Fc variant and the second Fc variant are selected from the group consisting of a Hole-modified Fc and a Knob-modified Fc, respectively.
  • the modified first Fc variant and second Fc variant contribute to the formation of the heterodimeric protein by the mode of action of "Knob/Hole".
  • the second Fc variant is a Hole-modified Fc; or when the second Fc variant is a Knob-modified Fc, the first Fc variant is a Hole-modified Fc.
  • an IL-15 heterodimeric protein wherein the sequence of the first Fc variant is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28 And SEQ ID NO: 29, and wherein the sequence of the second Fc variant is selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29.
  • Protein (I) and protein ( ⁇ ) by SEQ ID NO: The Fc variant represented by 26 and SEQ ID NO: 27 or SEQ ID NO: 28 and SEQ ID NO: 29 forms a heterodimer in the mode of action of "Knob/Hole".
  • the first Fc variant sequence is SEQ ID NO: 26
  • the second Fc variant sequence is SEQ ID NO: 27
  • the first Fc variant The sequence is SEQ ID NO:27.
  • an IL-15 heterodimeric protein wherein the sequence of said protein (I) is selected from the group consisting of SEQ ID NOs: 14-17, preferably SEQ ID NO: 14.
  • an IL-15 heterodimeric protein wherein the sequence of the protein ( ⁇ ) is selected from the group consisting of SEQ ID NOs: 18-25, 34-37, preferably selected from the group consisting of SEQ ID NO : 23, 34-37, more preferably selected from the group consisting of SEQ ID NOs: 34-37.
  • an IL-15 heterodimeric protein wherein the sequence of the protein (I) is selected from the group consisting of SEQ ID NOs: 30-31; the sequence of the protein ( ⁇ ) is selected from the group consisting of SEQ ID NOs: 32-33.
  • the IL-15 heterodimeric protein of the present invention is selected from the following dimeric proteins 3-17, wherein the dimeric proteins 3-17 are respectively composed of corresponding proteins (I) and proteins ( ⁇ ) Reorganized:
  • the invention also relates to a nucleic acid encoding an IL-15 heterodimeric protein as described above.
  • the invention also relates to a DNA vector comprising a nucleic acid as described above.
  • the invention also relates to a host cell transfected with the DNA vector of the invention.
  • the present invention also relates to a method of producing an IL-15 heterodimeric protein as described above, the method comprising: culturing the culture under conditions sufficient to express an IL-15 heterodimeric protein as described above
  • the host cell of the invention expresses and purifies the IL-15 heterodimeric protein.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a heterodimeric protein of IL-15 according to the invention and a pharmaceutically acceptable excipient, dilution or carrier.
  • the invention also relates to a targeting protein molecule comprising an IL-15 heterodimeric protein structure according to the invention.
  • the invention also relates to a method for stimulating or inhibiting an immune response in a mammal comprising: The mammal is administered an effective amount of an IL-15 heterodimeric protein according to the invention, or a pharmaceutical composition according to the invention, or a targeting protein molecule according to the invention.
  • the invention further relates to an IL-15 heterodimeric protein according to the invention, or a pharmaceutical composition according to the invention, or a targeting protein molecule according to the invention, prepared for the treatment of IL Use in a medicament for -15 mediated diseases or conditions; wherein the diseases are infectious diseases, cancer, blood diseases, and autoimmune diseases.
  • the cancer is selected from the group consisting of melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, solid tumor, liver cancer, lung cancer, stomach cancer, breast cancer; the infectious disease is selected from the group consisting of smallpox virus infection, HIV infection, and bacteria.
  • the blood disease is selected from the group consisting of anemia, acute myeloid leukemia, myelodysplastic syndrome, T-cell large granular lymphocytic leukemia;
  • the autoimmune disease is selected from multiple sclerosis Symptoms, psoriasis, rheumatoid arthritis, inflammatory diseases, gastritis, mucositis.
  • the drug is an IL-15 heterodimeric protein according to the present invention, or a combination of the pharmaceutical composition of the present invention and a small molecule inhibitor or an antibody drug; the small molecule inhibition
  • the agent is preferably an alkylating agent;
  • the antibody drug is preferably a monoclonal antibody drug, more preferably an anti-CD20, PD1, PDL1, Her2 antibody.
  • the medicament according to the invention is used in combination with a therapeutically effective amount of a medicament selected from the group consisting of temozolomide, doxorubicin, paclitaxel, cisplatin, carboplatin, dacarbazine, topotecan, irinotecan, gemcitabine And bevacizumab.
  • the invention further relates to an IL-15 heterodimeric protein according to the invention, or a pharmaceutical composition according to the invention, or a targeting protein molecule according to the invention, in cellular immunotherapy Uses, especially in the treatment of DC, CIK, DC-CIK, ECIK, NK, CAS-T, BiAb-T, TCR-T, CAR-T tumor immunocytes.
  • Tumor immunotherapy is a hot spot in the field of cancer treatment, and it is the fourth tumor treatment mode after surgery, chemotherapy and radiotherapy.
  • the purpose of tumor immunology treatment is to stimulate or modulate the immune system of the motivational body, enhance the anti-tumor immunity of the tumor microenvironment, and thereby control and kill tumor cells. It may be the most effective and safest way to treat tumors.
  • the immune escape mechanism of tumors is to maintain or promote the growth of tumors by using the tumor itself to inhibit the immune system.
  • Tumor immunotherapy is to maximize the patient's own immune system response to the tumor. It not only activates the original immune system response in the body, but also maintains the duration of the immune system response and the intensity of the response. This is the immunotherapy tumor. key.
  • Cytokine immunotherapy is developed with the production of high purity or recombinant cytokines.
  • the principle is that after using certain cytokines to inject, the function of one or more immune cells can be adjusted and enhanced, and a stronger anti-tumor immunity effect is exerted.
  • the present invention also relates to a method of treating or preventing a disease in which a cell expresses a disease-associated antigen, the method comprising: administering to a patient an IL-15 heterodimeric protein according to the present invention, Or a pharmaceutical composition according to the present invention, or a targeting protein molecule according to the present invention; forming a specificity between the cell expressing the disease-associated antigen and the immune cell expressing IL-15R ⁇ sufficient to activate the immune cell a sex binding complex; and killing the cell expressing the disease-associated antigen by the immune cell.
  • the cell expressing the disease-associated antigen is preferably a tumor cell or a virus-infected cell.
  • the immune cells described therein are preferably T-cells, LAK cells or NK cells.
  • the diseases described therein are infectious diseases, cancers, blood diseases and autoimmune diseases.
  • the cancer is selected from the group consisting of melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, solid tumor;
  • the infectious disease is selected from the group consisting of smallpox virus infection, HIV infection, bacterial infection, fungal infection;
  • the disease is selected from the group consisting of anemia, acute myeloid leukemia, myelodysplastic syndrome, T-cell large granular lymphocytic leukemia;
  • the autoimmune disease is selected from the group consisting of multiple sclerosis, psoriasis, rheumatoid arthritis, inflammation sexual diseases, gastritis, mucositis.
  • the invention also relates to a method of treating or preventing a disease comprising administering to a patient an IL-15 heterodimeric protein according to the invention, or a pharmaceutical composition according to the invention, or as according to the invention
  • the targeted protein molecule, and the combined administration of other drugs such as small molecule inhibitors or antibody drugs
  • the small molecule inhibitor is preferably an alkylating agent
  • the antibody drug is preferably a monoclonal antibody drug, more preferably Anti-CD20, PD1, PDL1, Her2 antibodies.
  • heterodimeric protein refers to a protein in which two different monomeric proteins are combined.
  • two different monomeric proteins each contain an Fc fragment or an Fc variant fragment, and form a heterodimeric protein by interaction of an Fc fragment or an Fc variant fragment.
  • the “interaction” between the first Fc variant and the second Fc variant in the present invention refers to the inter-Fc variant interaction.
  • Fc variant refers to a change in Fc structure or function caused by the presence of one or more amino acid substitutions, insertions or deletions at a suitable site on the Fc.
  • Interacting Fc variant refers to a space-filling effect, electrostatic guidance, hydrogen bonding, hydrophobic interaction, etc., between Fc variants designed by mutation.
  • the inter-Fc variant interaction contributes to the formation of a stable heterodimeric protein.
  • the preferred mutation is designed as a mutation design in the form of "Knob-into-Hole".
  • the "monomer protein” (i.e., protein (I), protein ( ⁇ )) constituting the heterodimeric protein in the present invention may be a fusion protein or a non-fusion protein.
  • the "fusion protein” as used in the present invention refers to a protein product obtained by expressing a recombinant gene under the control of the same regulatory sequence by linking the coding regions of two or more genes by genetic recombination, chemical methods or other appropriate methods.
  • the protein (I) is a fusion protein obtained by recombinant expression of IL-15 or a variant thereof and an Fc variant gene; the protein ( ⁇ ) can be recombinantly expressed by the IL-15R ⁇ and the Fc variant gene. Fusion protein.
  • the coding regions of two or more genes may be fused at one or several positions by a sequence encoding a peptide linker. Peptide linkers can also be used to construct fusion proteins of the invention.
  • the "IL-15” or "IL-15 peptide” of the present invention may be any IL-15 (interleukin 15) or a mutant thereof, such as human IL-15 or non-human mammal IL-15 or non-mammal IL-15.
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens and the like.
  • Preferred human interleukin 15 mature molecule see database UniProtKB, accession number P40933, 49-162aa.
  • the term "IL-15 variant” refers to an increase or decrease in affinity between IL-15 and its receptor obtained by one or more amino acid substitutions, additions or deletions, or an increase or decrease in the activity of T cells or NK cells. Mutant molecule.
  • the "IL-15R ⁇ ” described in the present invention may be IL-15Ra of any species or a functional fragment thereof, such as human IL-15Ra or non-human mammal IL-15Ra or non-mammalian IL-15Ra.
  • exemplary non-human mammals such as pigs, rabbits, monkeys, orangutans, mice, etc., non-mammals such as chickens and the like.
  • Preferred is human IL-15Ra, more preferably human interleukin 15 receptor alpha extracellular domain fragment, abbreviated as IL-15R ⁇ ECD (SEQ ID NO: 2), see database UniProtKB, accession number Q13261, 31-205aa.
  • IL-15R ⁇ variant refers to a functional mutant, preferably a human IL15Ra molecule, which has the ability to bind to its ligand molecule, such as IL15, by one or more amino acid deletions, insertions or substitutions on IL-15Ra.
  • the shortened form of the human IL-15R ⁇ extracellular domain fragment ie, a molecule having human interleukin 15 receptor alpha activity obtained by one or more amino acid deletion mutations from the C-terminus of the extracellular domain fragment, preferably retains 65-120 amino acids A deletion mutant form, more preferably a deletion mutant shortening form retaining 65-102 amino acids, such as IL-15R ⁇ -sushi (77) (SEQ ID NO: 3), IL-15R ⁇ -sushi (65) (SEQ ID NO: 4) .
  • immunoglobulin Fc region refers to an immunoglobulin chain constant region, particularly a carboxy terminus or a portion thereof of an immunoglobulin heavy chain constant region, free of antigen binding activity, and is an antibody molecule that interacts with effector molecules and cells.
  • an immunoglobulin Fc region can include a combination of two or more domains of heavy chains CH1, CH2, CH3, CH4 and an immunoglobulin hinge region.
  • Fc may be derived from a different species, preferably a human immunoglobulin.
  • immunoglobulins can be divided into different classes, mainly including five types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these may be further divided into subclasses (isotypes) such as IgG-1, IgG-2, IgG-3, IgG-4; IgA-1 and IgA-2.
  • the "Fc region” preferably includes at least one immunoglobulin hinge region, as well as the CH2 and CH3 regions of IgG. More preferably, it comprises a CH2 domain of IgG1, a CH3 domain and an immunoglobulin hinge region, and the starting amino acid position of the hinge region can be varied.
  • Mutation design techniques for Fc variants have been widely used in the art to prepare bispecific antibodies or heterodimeric Fc fusion protein forms. Representative examples are the "Knob-into-Hole" format proposed by Cater et al. (Protein Engineering vol. 9 no. 7 pp. 617-621, 1996); Amgen technicians use Electrostatic Steering to form Fc-containing isoforms. Source dimer form (US 20100286374A1); Jonathan H. Davis et al. (Protein Engineering, Design & Selection pp.
  • the Knob/Hole structure on the Fc variant fragment of the present invention means that each of the two Fc fragments is mutated, and the mutation can be combined by the "Knob-into-Hole" form.
  • the site mutation modification is preferably performed on the Fc region using the "knob-into-hole" model of Cater et al., such that the first Fc variant and the second Fc variant obtained can be in the form of "knob-into-hole" Together, a heterodimer is formed. Selection of a particular immunoglobulin Fc region from a particular immunoglobulin class and subclass is well within the purview of those skilled in the art.
  • the Fc region of human antibodies IgG1, IgG2, IgG3, IgG4 is preferred, and the Fc region of human antibody IgG1 is more preferred.
  • One of the first Fc variants or the second Fc variant is randomly selected to be a mutation of the knob, and the other is a mutation of the hole.
  • the first Fc variant is a mutation of a knob, such as the sequence SEQ ID NO: 26;
  • the second Fc variant is a mutation in a hole, such as the sequence SEQ ID NO 27.
  • Linker is used in the present invention to bind an IL-15 or IL-15R ⁇ with an Fc variant to ensure proper folding and stability of the protein.
  • the "linker peptide” of the present invention is preferably (GGGGS) n , wherein n may be 0, 1, 2, 3, 4, 5 or more, preferably n is 2-4. If the linker peptide sequence is too short, it may affect the folding of the higher structure of the two proteins, thereby interfering with each other; if the linker peptide sequence is too long, it involves the problem of immunogenicity, since the linker peptide sequence itself is a new antigen.
  • heterodimeric protein described in the present invention is preferably a product in which a gene is co-expressed.
  • a gene for example, co-expression in prokaryotic cells in Enterobacter; or co-expression in eukaryotic cells such as 293 and CHO.
  • Codon as used in the present invention means that a plurality of genes are expressed together in one cell, and their products appear at the same time. These genes may be present simultaneously and separately or collectively. In the present invention, it is preferred to co-express two genes in one eukaryotic cell. Co-expression of the resulting gene expression product facilitates efficient and simple formation of a complex; in the present invention, it facilitates the formation of a heterodimeric protein.
  • the immunoglobulin of the present invention is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the immunoglobulin heavy chain constant region has different amino acid composition and arrangement order, so its antigenicity is also different. Accordingly, immunoglobulins can be classified into five classes, or isoforms of immunoglobulins, i.e., IgM, IgD, IgG, IgA, and IgE.
  • the same type of Ig can be divided into different subclasses according to the difference in the amino acid composition of the hinge region and the number and position of heavy chain disulfide bonds. For example, IgG can be classified into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are divided into kappa or lambda chains by the constant region.
  • a targeted protein molecule which refers to a class of protein molecules containing a fragment or region capable of interacting with other protein molecules, by designing a unique molecule in an immune cytokine such as IL5, IL2, or the like, or such a cytokine, such as this application
  • the molecular design is based on the introduction of targeting peptides such as antibody fragments, ScFv or binding peptides of certain cell surface molecules.
  • an antibody interacts with an antigen, or a ligand interacts with a receptor, so that when the molecule enters the body, it can preferentially be enriched in a specific tissue, organ or body part by targeting to exert its biological function.
  • this approach can be rationally derivatized in the art to produce a series of molecules.
  • administering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, animal or subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment when applied to a human, animal or subject, or cell, tissue or organ, includes an IL-15 agonist or an IL-15 antagonist and a human or animal, subject, cell, tissue, physiological compartment Or contact with physiological fluids.
  • Treatment of cells also includes the case where an IL-15 agonist or an IL-15 antagonist, for example, contacts the IL-15 receptor in a fluid phase or a colloidal phase, and includes the case where the agonist or antagonist does not contact the cell or receptor. .
  • Treatment means administration of a therapeutic agent for internal or external use to a patient, such as a composition comprising an IL-15 heterodimeric protein of the invention.
  • the patient has one or more diseases or symptoms.
  • the therapeutic agents are known to have a therapeutic effect on these diseases or conditions.
  • the therapeutic agent is administered in an amount effective to alleviate one or more diseases or conditions in the patient or population being treated, whether by inducing the degradation of such disease or condition or inhibiting the progression of such disease or condition to any clinically measurable degree.
  • the amount of therapeutic agent (also referred to as “therapeutically effective amount”) effective to alleviate any particular disease or condition can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Immunotapéutica disorder or “immune disorder” includes, for example, pathological inflammation, inflammatory conditions, and autoimmune diseases or diseases. “Immune disorder” also refers to infections, persistent infections, and proliferative disorders such as cancer, tumors, and angiogenesis. “Cancerous conditions” include, for example, cancer, cancer cells, tumors, angiogenesis, and precancerous conditions, such as dysplasia.
  • PCR polymerase chain reaction
  • “Optional” or “optionally” means that the subsequently described event or environment may, but need not, occur, including where the event or environment occurs or does not occur.
  • “optionally comprising 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable region may, but need not, be present; when present, it may be one, two or three.
  • “Pharmaceutical composition” means a mixture comprising one or more of the compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components, as well as other components such as physiological/pharmaceutically acceptable carriers. And excipients.
  • the purpose of the pharmaceutical composition is to promote the administration of the organism, which facilitates the absorption of the active ingredient and thereby exerts biological activity.
  • the step of transforming a host cell with recombinant DNA as described in the present invention can be carried out by a conventional technique well known to those skilled in the art.
  • the obtained transformant can be cultured by a conventional method, and the polypeptide encoded by the gene of the present invention is expressed.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the host cell is cultured under conditions suitable for the growth of the host cell.
  • Figure 1 Relationship between dosing time and molar concentration of samples in serum.
  • Test Example 3 Number of metastatic tumor masses in mice administered in a lung metastasis model. * in the figure indicates: p ⁇ 0.05, vsPBS; ** indicates: p ⁇ 0.01, vsPBS; ## indicates: p ⁇ 0.01, vs IL-15.
  • Figure 7 Efficacy of different drug-administered proteins on HCT-116+PBMC SCID xenografts, tumor weight on day 28. * in the figure indicates: p ⁇ 0.05, vs PBMC.
  • FIG. 8 Comparison of the number of lung metastasis tumor spots in mice after administration of dimeric protein 17, positive control (IL-15) and negative control (PBS) in mice, *: p ⁇ 0.05, **: p ⁇ 0.01, vs PBS.
  • FIG. 1 Comparison of relative lung weight (lung weight/body weight) of mice after administration of dimeric protein 17, positive control (IL-15) and negative control (PBS) to mice.
  • IL-15 positive control
  • PBS negative control
  • FIG. 1 Comparison of mouse body weights of mice after administration of dimeric protein 17, positive control (IL-15) and negative control (PBS) to mice.
  • IL-15 positive control
  • PBS negative control
  • the heterodimeric protein provided by the present invention is formed by the combination of protein (I) and protein (I), which is a fusion of IL-15 or a variant thereof and the first Fc variant.
  • the body binds to the second Fc variant in the form of Knob/Hole.
  • IL-15 as used in the examples of the present invention refers to a human interleukin 15 mature molecule (SEQ ID NO: 1) or a variant thereof.
  • the IL-15R ⁇ ECD used in the examples of the present invention refers to a human interleukin 15 receptor alpha extracellular domain fragment (SEQ ID NO: 2); variants thereof are preferably in a shortened form, such as IL-15R ⁇ -sushi (77) (SEQ ID NO: 3), IL-15R ⁇ -sushi (65) (SEQ ID NO: 4).
  • the Fc fragment portion used in the examples of the present invention may be an Fc fragment of human antibody IgG1, IgG2, IgG3, IgG4, preferably an Fc fragment of human IgG1.
  • the first Fc variant or the second Fc variant in the present invention is preferably subjected to a mutation in the Knob form (SEQ ID NO: 26), or a mutation in the Hol form (SEQ ID NO: 27).
  • a protein is formed between the protein (I) and the protein ( ⁇ ) by a Knob/Hole structure between the first Fc variant and the second Fc variant, preferably by the first The "Knob-into-Hole" mode of action between the Fc variant and the second Fc variant forms a heterodimer.
  • a dimer containing only a single IL-15 effector molecule can also be fused to the first Fc variant by IL-15, and the first Fc variant and the corresponding second Fc variant are Knob-into-Hole
  • the mode of action forms, as in the heterodimeric protein 3 of the invention.
  • the present invention fused IL-15 or a variant thereof to a first Fc fragment or a first Fc variant via a linker peptide to form a fusion protein, and the present invention passes IL-15R ⁇ ECD or a variant thereof through a linker peptide and a second Fc fragment Or the second Fc variant is fused to form a fusion protein, wherein the order of linkage of each protein component is not limited; the linker peptide may be a soft linking peptide commonly used in the field, preferably (GGGGS)n, wherein n is selected from 1-10, It is preferably selected from 1-5, most preferably 2.
  • IL-15 protein sequence 1: (amino acid sequence of human interleukin 15, also a reference IL-15 sequence)
  • IL-15R ⁇ ECD protein sequence 2: (the amino acid sequence of the extracellular domain of human interleukin 15 receptor alpha, also used to fuse with Fc to form the part of IL-15Ra in molecule H)
  • IL-15R ⁇ -sushi (77) (protein sequence 3): (a domain that can maintain more than 90% of the binding activity of the human interleukin 15 receptor alpha ectodomain, called the sushi domain plus a small stretch of the linker peptide, belonging to the shortened form IL-15R ⁇ )
  • IL-15R ⁇ -sushi (65) (protein sequence 4): (a domain in which the human interleukin 15 receptor alpha extracellular domain is capable of retaining more than 90% of the binding activity, the so-called sushi domain belongs to the shortened form of IL-15Ra)
  • IL15Ra-sushi (86) (protein sequence 6):
  • IL-15-Fc protein sequence 8: (a fusion protein formed by the human interleukin 15 molecule linked to a human IgG1-Fc sequence by a linker peptide, expressed as a bivalent homodimer, where the IL-15 molecule is At the N-terminus of the protein)
  • Fc-IL-15 protein sequence 9): (a fusion protein formed by a human interleukin 15 molecule linked to a human IgG1-Fc sequence by a linker peptide, expressed as a bivalent homodimer, wherein the IL-15 molecule is At the C-terminus of the protein)
  • IL-15R ⁇ ECD-Fc protein sequence 10: (a fusion protein formed by the human interleukin 15 receptor alpha extracellular domain linked to a human IgG1-Fc sequence by a linker peptide, expressed as a bivalent homodimer, wherein IL The -15R ⁇ -ECD molecule is at the N-terminus of the protein)
  • Fc-IL-15R ⁇ ECD protein sequence 11: (a fusion protein formed by the human interleukin 15 receptor alpha extracellular domain linked to a human IgG1-Fc sequence by a linker peptide, expressed as a bivalent homodimer, wherein IL -15R ⁇ ECD molecule is at the C-terminus of the protein)
  • IL-15R ⁇ -sushi(77)-Fc protein sequence 12: (Sushi (77) fragment consisting of the sushi domain of human interleukin 15 receptor alpha extracellular domain plus a ligation peptide via a linker peptide to human IgG1-Fc a fusion protein formed by sequence ligation, the sushi (77) fragment is located at the N-terminus)
  • Fc-IL-15R ⁇ -sushi (77) (protein sequence 13): (Sushi+ fragment consisting of the sushi domain of the human interleukin 15 receptor alpha extracellular domain plus a ligation peptide is formed by ligation of a linker peptide to the human IgG1-Fc sequence. Fusion protein, sushi+ fragment located at the C-terminus)
  • IL-15-Fc-Knob protein sequence 14: (The Fc portion of SEQ ID NO: 8 is mutated to the Knob form and can be paired with the Hol form of another fusion molecule)
  • IL-15-Fc-Hole protein sequence 15: (The Fc portion of the above sequence 8 is mutated to a Hole form and can be paired with the Knob form of another fusion molecule)
  • Fc-Knob-IL-15 protein sequence 16: (The Fc portion of the above sequence 9 is mutated to the Knob form and can be paired with the Hol form of another fusion molecule)
  • Fc-Hole-IL-15 protein sequence 17: (The Fc portion of the above sequence 9 was mutated to a Hole form and can be paired with the Knob form of another fusion molecule)
  • IL-15R ⁇ ECD-Fc-Knob protein sequence 18: (The Fc portion of the above sequence 10 is mutated to the Knob form and can be paired with the Hole form of another fusion molecule)
  • IL-15R ⁇ ECD-Fc-Hole protein sequence 19: (The Fc portion of the above sequence 10 is mutated to a Hole form and can be paired with the Knob form of another fusion molecule)
  • Fc-Knob-IL-15R ⁇ ECD protein sequence 20: (The Fc portion of the above sequence 11 is mutated to the Knob form and can be paired with the Hol form of another fusion molecule)
  • Fc-Hole-IL-15R ⁇ ECD protein sequence 21: (The Fc portion of the above sequence 11 is mutated to a Holic form and can be paired with the Knob form of another fusion molecule)
  • IL-15R ⁇ -sushi(77)-Fc-Knob protein sequence 22: (The Fc portion of protein sequence 12 is mutated to the Knob form and can be paired with the Hol form of another fusion molecule)
  • IL-15R ⁇ -sushi(77)-Fc-Hole protein sequence 23: (The Fc portion of protein sequence 12 is mutated to a Hole form and can be paired with the Knob form of another fusion molecule)
  • Fc-Knob-IL-15R ⁇ -sushi (77) (protein sequence 24): (Fc portion of protein sequence 13) Amino acid mutations into the Knob form can be paired with the Hol form of another fusion molecule)
  • Fc-Hole-IL-15R ⁇ -sushi (77) (protein sequence 25): (The Fc portion of protein sequence 13 is mutated to a Hole form and can be paired with the Knob form of another fusion molecule)
  • Fc-Knob protein sequence 26: (Knob mutant form of human IgG1-Fc portion, which can be paired with IL-15-Fc-Hole/Fc-IL-15-Hole)
  • Fc-Hole protein sequence 27: (Hole mutant form of human IgG1-Fc portion, which can be paired with IL-15-Fc-Knob/Fc-IL-15-Knob
  • Fc-Knob (M) protein sequence 28
  • Fc-Hole (M) Another way of Fc mutation, which can be paired with Fc-Hole (M) to form a heterodimer.
  • Fc-Hole (M) protein sequence 29: another way of Fc mutation, and Fc-Knob (M) Pairing forms a heterodimer.
  • Fc-Knob(M)-IL-15 protein sequence 30 (different from the Knob mutation site, another heterodimer mutation mode)
  • IL-15-Fc-Knob (protein sequence 31) (different from the Knob mutation site, another heterodimer mutation mode)
  • Fc-Hole(M)-IL-15R ⁇ -sushi(65) protein sequence 32 (different to the hetero-cleavage site, another heterodimer mutation)
  • IL-15R ⁇ -sushi(73)-Fc-Hole protein sequence 34: (sushi (73) refers to a shortened form of IL15R ⁇ containing a sushi domain of 73 amino acids in length)
  • IL-15R ⁇ -sushi(65)-Fc-Hole protein sequence 35: (sushi (65) refers to the sushi domain of 65 amino acids in length)
  • IL-15R ⁇ -sushi(86)-Fc-Hole protein sequence 36: (sushi (86) refers to a shortened form of IL-15R ⁇ containing a sushi domain of 86 amino acids in length)
  • IL-15R ⁇ -sushi(102)-Fc-Hole protein sequence 37: (sushi (102) refers to a shortened form of IL-15R ⁇ containing a sushi domain of 102 amino acids in length)
  • Eukaryotic expression vector pcDNA3.1(+) (Life technologies, Cat. No. V790-20);
  • IL-15 DNA sequence 1
  • IL-15R ⁇ ECD DNA sequence 2
  • IgG1Fc DNA sequence 3
  • DNA fragment synthesized by gene synthesis company (Jin Weizhi, Suzhou);
  • Primer DNA fragment synthesized by gene synthesis company (Jin Weizhi, Suzhou).
  • Fragment splicing was carried out by a conventional PCR method.
  • IL-15-Fc fragment Overlap PCR was performed in the order of IL-15, the linker peptide, and Fc, and the three DNA fragments were spliced into an IL-15-Fc fragment (DNA sequence 4, SEQ ID NO: 42).
  • IL-15R ⁇ ECD-Fc fragment The overlapped PCR was carried out in the order of IL-15R ⁇ ECD, the linker peptide, and the Fc, and the three DNA fragments were spliced into an IL-15R ⁇ ECD-Fc fragment (DNA sequence 5, SEQ ID NO: 43).
  • Fc-IL-15 fragment Overlap PCR was carried out in the order of Fc, linker peptide, IL-15, and the three DNA fragments were spliced into an Fc-IL-15 fragment (DNA sequence 6, SEQ ID NO: 44).
  • Fc-IL-15R ⁇ ECD fragment Overlap PCR was carried out in the order of Fc, linker peptide, IL-15R ⁇ ECD, and three DNA fragments were spliced into an Fc-IL-15R ⁇ ECD fragment (DNA sequence 7, SEQ ID NO: 45).
  • IL-15-Fc-Knob fragment Overlap PCR was performed in the order of IL-15, linker peptide, Fc-Knob, and three DNA fragments were spliced into IL-15-Fc-Knob fragment (DNA sequence 8, SEQ ID NO :46).
  • IL-15-Fc-Hole fragment Overlap PCR was performed in the order of IL-15, linker peptide, Fc-Hole, and the three DNA fragments were spliced into an IL-15-Fc-Hole fragment (DNA sequence 9, SEQ ID NO :47).
  • Fc-Knob-IL-15 fragment Fc-Knob, ligation peptide, IL-15 sequence, overlap PCR, splicing three DNA fragments into Fc-Knob-IL-15 fragment (DNA sequence 10, SEQ ID NO :48).
  • Fc-Hole-IL-15 fragment Fc-Hole, ligation peptide, IL-15 sequence, overlap PCR, splicing three DNA fragments into Fc-Hole-IL-15 fragment (DNA sequence 11, SEQ ID NO :49).
  • IL-15R ⁇ ECD-Fc-Knob fragment in the order of IL-15R ⁇ ECD, ligation peptide, Fc-Knob Three copies of the DNA fragment were spliced into an IL-15R ⁇ ECD-Fc-Knob fragment (DNA sequence 12, SEQ ID NO: 50) by overlapping PCR.
  • IL-15R ⁇ ECD-Fc-Hole fragment Overlap PCR was performed in the order of IL-15R ⁇ ECD, ligation peptide, Fc-Hole, and three DNA fragments were spliced into IL-15R ⁇ ECD-Fc-Hole fragment (DNA sequence 13, SEQ ID NO :51).
  • Fc-Knob-IL-15R ⁇ ECD fragment Fc-Knob, ligation peptide, IL-15R ⁇ ECD sequence, overlap PCR, splicing three DNA fragments into Fc-Knob-IL-15R ⁇ ECD fragment (DNA sequence 14, SEQ ID NO :52).
  • Fc-Hole-IL-15R ⁇ ECD fragment Fc-Hole, ligation peptide, IL-15R ⁇ ECD sequence, overlap PCR, splicing three DNA fragments into Fc-Hole-IL-15R ⁇ ECD fragment (DNA sequence 15, SEQ ID NO :53).
  • Fc-Knob fragment DNA sequence 16, SEQ ID NO:54.
  • Fc-Hole fragment DNA sequence 17, SEQ ID NO:55.
  • Fc-Knob (M)-IL-15 DNA sequence 18, the nucleotide sequence encoding the protein sequence 30 precursor, SEQ ID NO:56.
  • IL-15-Fc-Knob DNA sequence 19 the nucleotide sequence encoding the precursor of protein sequence 31, SEQ ID NO:57.
  • IL15R ⁇ -sushi(65)-Fc-Hole(M) DNA sequence 21, the nucleotide sequence encoding the precursor of protein sequence 33, SEQ ID NO:59.
  • IL-15R ⁇ -sushi(65)-Fc-Hole DNA sequence 23, the nucleotide sequence encoding the 35 sequence of the protein sequence, SEQ ID NO:61.
  • IL-15R ⁇ -sushi(102)-Fc-Hole DNA sequence 25 the nucleotide sequence encoding the protein sequence 37 precursor, SEQ ID NO:63.
  • a restriction endonuclease KpnI site, a Kozak sequence, and a signal peptide sequence were introduced at the 5' end of the gene fragment by a PCR method.
  • the sequence between the KpnI site and the gene fragment is (SEQ ID NO: 38):
  • the above gene fragments were inserted into the vector pcDNA3.1(+) using KpnI and NotI restriction enzyme sites, respectively, and constructed into expression vectors such as pcDNA3.1-IL-15-Fc, pcDNA3.1-IL-15R ⁇ ECD-Fc, pcDNA3.1. -Fc, pcDNA3.1-Fc-IL-15, pcDNA3.1-Fc-IL-15R ⁇ ECD, etc., to obtain the corresponding expression plasmid.
  • the site-directed mutagenesis process uses a KOD kit (TOYOBO Cat.KOD-201) 25 ⁇ L system: 2.5 ⁇ L 10 ⁇ KOD buffer, 2.5 ⁇ L 2 mM dNTPs, 1 ⁇ L primer 1 (10 ⁇ M), 1 ⁇ L primer 2 (10 ⁇ M), 0.5 ⁇ L KOD plus, 1 ⁇ L 25 mM MgSO 4 , 16 ⁇ L ddH 2 O.
  • the synthesis procedure was 94 ° C for 2 minutes, 94 ° C for 30 seconds, 55 ° C for 30 seconds, 68 ° C for 11 minutes, amplification for 25 cycles, and then 68 ° C for 11 minutes to end the PCR amplification procedure.
  • the protein 3 according to the embodiment of the present invention is expressed by an expression vector containing DNA sequence 8 (SEQ ID NO: 46) and DNA sequence 17 (SEQ ID NO: 55); and protein 7 is composed of DNA sequence 8 (SEQ ID NO: 46) And the expression vector of DNA sequence 13 (SEQ ID NO: 51) was expressed. Expression of other examples was obtained by co-expression of an expression vector of the DNA sequence constituting the protein.
  • the following sequence is the DNA sequence used to construct the vector, and the underlined portion is the signal peptide DNA sequence, the underlined portion is the peptide linker DNA sequence, and the double-lined portion is the DNA sequence of the Fc for Knob/Hole mutation.
  • DNA sequence 1 (IL-15, nucleotide sequence of human interleukin 15)
  • DNA sequence 2 (IL-15R ⁇ ECD, the extracellular domain nucleotide sequence of human interleukin 15 receptor alpha)
  • DNA sequence 3 (Fc, Fc nucleotide sequence of human IgG)
  • DNA sequence 4 (IL-15-Fc, a nucleotide sequence encoding a protein sequence 5 precursor)
  • DNA sequence 5 (IL-15R ⁇ ECD-Fc, a nucleotide sequence encoding a protein sequence 7 precursor)
  • DNA sequence 6 (Fc-IL-15, a nucleotide sequence encoding a protein sequence 6 precursor)
  • DNA sequence 7 (Fc-IL-15R ⁇ ECD, a nucleotide sequence encoding a protein sequence 8 precursor)
  • DNA sequence 8 (IL-15-Fc-Knob, a nucleotide sequence encoding a protein sequence 11 precursor)
  • DNA sequence 9 (IL-15-Fc-Hole, a nucleotide sequence encoding a protein sequence 12 precursor)
  • DNA sequence 10 (Fc-Knob-IL-15, a nucleotide sequence encoding a protein sequence 13 precursor)
  • DNA sequence 11 (Fc-Hole-IL-15, a nucleotide sequence encoding a protein sequence 14 precursor)
  • DNA sequence 12 (IL-15R ⁇ ECD-Fc-Knob, a nucleotide sequence encoding a protein sequence 15 precursor)
  • DNA sequence 13 (IL-15R ⁇ ECD-Fc-Hole, a nucleotide sequence encoding a protein sequence 16 precursor)
  • DNA sequence 14 (Fc-Knob-IL-15R ⁇ ECD, a nucleotide sequence encoding a protein sequence 17 precursor)
  • DNA sequence 15 (Fc-Hole-IL-15R ⁇ ECD, a nucleotide sequence encoding a protein sequence 18 precursor)
  • DNA sequence 16 (Fc-Knob, a nucleotide sequence encoding a protein sequence 23 precursor)
  • DNA sequence 17 (Fc-Hole, a nucleotide sequence encoding a protein sequence 24 precursor)
  • DNA sequence 18 (Fc-Knob (M)-IL-15, a nucleotide sequence encoding a protein sequence 30 precursor):
  • DNA sequence 19 (IL-15-Fc-Knob (M), a nucleotide sequence encoding a protein sequence 31 precursor):
  • DNA sequence 20 (Fc-Hole (M)-IL-15R ⁇ -sushi (65), a nucleotide sequence encoding a protein sequence 32 precursor):
  • DNA sequence 21 (IL15R ⁇ -sushi(65)-Fc-Hole(M), a nucleotide sequence encoding a protein sequence 33 precursor):
  • DNA sequence 22 (IL-15R ⁇ -sushi(73)-Fc-Hole, a nucleotide sequence encoding a protein sequence 34 precursor):
  • DNA sequence 23 (IL-15R ⁇ -sushi(65)-Fc-Hole, a nucleotide sequence encoding a protein sequence 35 precursor):
  • DNA sequence 24 (IL-15R ⁇ -sushi(86)-Fc-Hole, a nucleotide sequence encoding a protein sequence 36 precursor):
  • DNA sequence 25 (IL-15R ⁇ -sushi(102)-Fc-Hole, a nucleotide sequence encoding a protein sequence 37 precursor):
  • the IL-15 protein was transiently transfected using FreeStyle 293 cells (GIBCO, Cat# R79007).
  • FreeStyle 293 cells were suspension cultured in a Freestyle 293 expression medium (GIBCO, Cat #12338018) with a final concentration of 1% Ultra Low IgG Fetal Bovine Serum (Ultra Low Immunoglobulin Fetal Bovine Serum, GIBCO, Cat #16250078).
  • the relevant expression plasmid and transfection reagent PEI (Polysciences, Cat#239662) in Example 1 was prepared, and the plasmid amount was 100 ug/100 ml of cells, and the mass ratio of plasmid to PEI was 1:2.
  • the cell density on the day of transfection was 1 ⁇ 10 6 /ml.
  • 1L of FreeStyle 293 was transfected into cells, and 50 ml of Opti-MEM (GIBCO, Cat#11058021) medium was mixed with the plasmid, allowed to stand for 5 min, and filtered; 50 ml of Opti-MEM medium was mixed with PEI and allowed to stand for 5 min. ,filter.
  • the plasmid and PEI were mixed and allowed to stand for 15 min.
  • the plasmid and PEI mixture were slowly added to the cells, and placed in a shaker incubator at 37 ° C, 8% CO 2 , 130 rpm. After 5 days, the supernatant was collected by centrifugation for protein purification.
  • the cell culture medium was centrifuged at a high speed, and the supernatant was taken, and affinity chromatography was performed using a Protein A column of GE. Chromatography was performed using 1 ⁇ PBS (pH 7.4) in the equilibration buffer. The cell supernatant was applied to the supernatant and washed with PBS until the UV was returned to the baseline. Then, the target protein was eluted with 0.1 M glycine (pH 3.0) in the elution buffer. , using Tris to adjust the pH to neutral preservation;
  • the product obtained by affinity chromatography is adjusted to a pH below or above 1-2 pH units, suitably diluted to control sample conductance below 5 ms/cm.
  • Ion exchange chromatography methods such as anion exchange or cation exchange conventional in the art are utilized using suitable corresponding pH buffers such as phosphate buffer, acetate buffer, and the like.
  • suitable corresponding pH buffers such as phosphate buffer, acetate buffer, and the like.
  • the gradient elution of NaCl under the corresponding pH conditions was carried out, and the collection tube in which the target protein was located was selected and stored according to SDS-PAGE.
  • the ion exchanged product is concentrated by ultrafiltration and subjected to size exclusion chromatography, such as separation using GE's Superdex 200 gel to remove possible aggregates and other components to obtain a high purity target product.
  • size exclusion chromatography such as separation using GE's Superdex 200 gel to remove possible aggregates and other components to obtain a high purity target product.
  • the resulting protein purity analysis can be analyzed by SDS-PAGE and SEC-HPLC detection. Protein concentration was determined by UV spectrophotometry.
  • the resulting protein sequence is as described in Example 1, and the specific heterodimeric protein is composed of one or two protein sequences selected from the above sequences, preferably heterologous dimers formed by pairing in the Knob/Hole form.
  • the molecules are purified; the bivalent protein can also be composed of the unmutated Fc chain region.
  • molecule 3 is formed by pairing the fusion protein IL-15-Fc-Knob and Fc-Hole (obtained by molecular purification after co-expression), and molecule 7 in the example passes the fusion proteins IL-15-Fc-Knob and IL-15R ⁇ ECD.
  • - Fc-Hole pair formation obtained by molecular purification after co-expression.
  • Fc-Knob (SEQ ID No. 26) 7 IL-15-Fc–Knob (SEQ ID No. 14) IL-15R ⁇ ECD-Fc-Hole (SEQ ID No.19) 8 IL-15-Fc-Hole (SEQ ID No. 15) IL-15R ⁇ ECD-Fc-Knob (SEQ ID No. 18) 9 Fc-Knob-IL-15 (SEQ ID No. 16) Fc-Hole-IL-15R ⁇ ECD (SEQ ID No. 21) 10 Fc-Hole-IL-15 (SEQ ID No. 17) Fc-Knob-IL-15R ⁇ ECD (SEQ ID No.
  • Table 1 shows the specific 17 dimeric proteins involved in the present invention, which are dimeric proteins 1-17, respectively, which are recombined from the corresponding proteins (I) and (II) in the table, respectively.
  • Fresh PBMC was cultured in RPMI1640 medium containing 10% FBS. After resuspending in the experiment, the density of the cells was adjusted to 5 ⁇ 10 5 /ml, 90 ul per well was added to the 96-well plate, and the sample was diluted with PBS to a certain multiple. Different concentration gradients, 10 ⁇ l per well in a 96-well plate, cultured at 37 ° C, 5% CO 2 incubator for 48 hours, 50 ⁇ l The Luminescent Cell Viability Assay kit detects cell proliferation. The results are shown in Table 2:
  • mice The pharmacokinetic changes of IL-15 heterodimeric proteins 3 and 7 in mice were evaluated.
  • test compound is a sample for loading
  • mice C57BL/6 mice, SPF, 15-16g, sputum, were purchased from Shanghai Xipuer Bikai Experimental Animal Co., Ltd.
  • mice Twenty-seven C57BL/6 mice were divided into 3 groups, 9 in each group, 3 in each cage, and 3 in each time point, and blood was collected by circulation. 2 ⁇ g of IL-15, 10 ⁇ g of dimeric protein 3 and 20 ⁇ g of dimeric protein 7 (IL-15, dimeric protein 3 and dimeric protein 7 equimolar, both 0.15 nmol) were intraperitoneally injected. Blood was taken at 30 min, 1, 2, 4, 8, 24, 48, 72, and 120 h after administration, and 50-100 ⁇ l of blood was taken from each eye. Serum was taken for human IL-15 ELISA.
  • dimeric protein 3 After equimolar administration of IL-15, dimeric protein 3, and dimeric protein 7, IL-15 reached a maximum within 30 minutes, then was rapidly metabolized over time and was completely metabolized by 24 hours.
  • Dimeric protein 3 reached a maximum 2 hours after administration and was then slowly metabolized over time until it was fully metabolized by 120 hours.
  • Dimeric protein 7 also reached a maximum 2 hours after administration, and was subsequently slowly metabolized over time, and a greater concentration of protein was detected by 120 hours.
  • the PK assay (Fig. 1) showed that the maximum protein molar concentration in the serum after administration of dimeric protein 3 and dimeric protein 7 was less than IL-15, but the retention time in serum exceeded IL-15, which had a significant long-term effect. effect.
  • IL-15 dimeric protein 3 and dimeric protein 7 were examined in a lung metastasis model, a nude mouse tumor bearing model, and a severe combined immunodeficiency NOD-SCID mouse model. The results are shown in the following test examples.
  • a mouse lung metastasis model was established using B16F10 cells to evaluate the effects of IL-15 drug administration on tumor metastasis and growth.
  • mice C57BL/6 mice, SPF, 10-16g, sputum, purchased from Shanghai Xipuer Bikai Experimental Animal Co., Ltd.
  • Dosing regimen 32 C57BL/6 mice were divided into 4 groups of 8 animals each. 1.5 ⁇ 10 5 B16F10 cells were injected through the tail vein, and PBS, 2 ⁇ g IL-15, 11 ⁇ g dimeric protein 3 and 14 ⁇ g dimeric protein 7 (IL-15, dimer) were intraperitoneally injected on days 1, 2 and 10. Protein 3 and dimeric protein 7 were equimolar, both 0.16 nmol).
  • mice On the 21st day, the mice were sacrificed, the lungs were removed, weighed, black tumor masses in the lungs were observed, photographed, fixed in neutral formaldehyde, and the number of black tumor masses was counted.
  • the IL-15 group had a large number of melanoma masses (157 ⁇ 20), which was about 90% of the PBS group;
  • the dimeric protein 3 administration group a small amount of metastatic melanoma mass (26 ⁇ 6) was observed in the lungs, which was about 15% in the PBS group; in the dimeric protein 7 administration group, more metastatic melanoma masses were observed in the lungs ( 83 ⁇ 28), which is about 49% of the PBS group.
  • the number of lung tumor blocks in the PBS group was significantly higher than that in the dimeric protein 3 and dimeric protein 7 groups, and there was no significant difference compared with the IL-15 group.
  • the number of lung tumor masses in the IL-15 group was significantly higher than that in the dimeric protein 3 group.
  • the number of lung tumor masses in the dimeric protein 7 group was significantly higher than that in the dimeric protein 3 group, as shown in Figure 2.
  • the relative weight of the lungs in the PBS group was significantly higher than that in the dimeric protein 3 and dimeric protein 7 administration groups, but there was no significant difference compared with the IL-15 group.
  • the relative weight of the lungs in the IL-15 group was significantly higher than that in the dimeric protein 3 and dimeric protein 7 groups, as shown in Figure 3.
  • a nude mouse tumor-bearing model was established using HCT-116 (human colon cancer) cells to evaluate the effect of IL-15 drug on tumor growth after administration.
  • BALB/cA-nude nude mice SPF, 16-20 g, sputum, were purchased from Shanghai Xipuer Bikai Experimental Animals Co., Ltd.
  • HCT-116 cells (5 ⁇ 10 6 /piece) were inoculated subcutaneously into the right flank of nude mice. The tumors were grown for 20 days and grown to 100 ⁇ 15 mm 3 . The animals were randomly divided into groups (d0), with 6 rats in each group.
  • IL-15 Intraperitoneal administration, once every 2 days, 3 times a week, IL-15 is 2 ⁇ g / only, dimeric protein 3 is 10 ⁇ g / only, dimeric protein 7 is 20 ⁇ g / only (IL-15 Dimer protein 3 and dimeric protein 7 are equimolar, both 0.15 nmol).
  • Tumor volume was measured 2-3 times per week (Fig. 4), body weight was weighed, and data was recorded. On the 27th day, he was killed and taken a tumor.
  • V 1/2 ⁇ L length ⁇ L short 2
  • Relative volume (RTV) V T /V 0
  • Tumor inhibition rate (%) (C RTV -T RTV ) / C RTV (%)
  • V 0, V T respectively start of the experiment and tumor volume at the end of the experiment.
  • C RTV and T RTV are the blank control group (Blank) at the end of the experiment and the relative tumor volume of the experimental group.
  • IL-15 protein The growth inhibitory effect of IL-15 protein on HCT-116 tumors is shown in Table 3 and Figure 4.
  • IL-15, 3 and 7 are administered in equimolar for 27 days, once every 2 days, IL-15, II. Both the aggregate protein 3 and the dimeric protein 7 group could inhibit the growth of HCT-16 cell xenografts, and the inhibition rates were 32%, 45%, and 20%, respectively.
  • Sex There was no death of the mice during the administration, and there was no significant decrease in body weight during the administration of the mice in each group, suggesting that there is no obvious side effect at the current dose.
  • Tumors of each group were weighed on the 27th day after administration, as shown in Fig. 5.
  • * indicates: p ⁇ 0.05 compared with the blank junction Blank, that is, the dimeric protein 3 group compared with the Blank group. The weight was significantly reduced, and the tumor weights of the IL-15 and dimeric protein 7 groups were comparable to those of the Blank group.
  • the anti-tumor effect of the three proteins is dimeric protein 3>IL-15>dimer protein 7.
  • HCT-116 cells and human peripheral blood mononuclear cells were mixed in vitro, and severe combined immunodeficient NOD-SCID mice were inoculated to evaluate the effect of IL-15 administration on tumor growth.
  • Nod-SCID mice theoretically lack T cells and NK cells themselves, so they can activate human PBMC after administration with IL-15.
  • Cells including T and NK cells) kill tumor cells and inhibit tumor cell growth.
  • the Nod-SCID mouse model is closer to the human immune system killing tumor cells.
  • IL-15 was purchased from Nearshore Protein Technology Co., Ltd.;
  • NOD SCID female mice were purchased from Beijing Weitong Lihua (batch number: 11400700006527), 6-8 weeks old, weighing about 20g, and 5 per drug group.
  • LSM Lymphocyte Separation Medium
  • centrifugation can precipitate red blood cells and multinucleated white blood cells and form a layer of mononuclear lymphocytes on LSM;
  • lymphocyte layer and the lower half of the LSM are transferred to another centrifuge tube.
  • the cells were washed with PBS buffer or RPMI-1640 medium, and the cells were resuspended in RPMI-1640 medium;
  • HCT-116 cells and PBMC cells were mixed uniformly in a ratio of 4:1, and mixed cells were subcutaneously inoculated into the right flank of SCID mice (HCT-116 cells: 5 ⁇ 10 6 /only, PBMC: 1.25 ⁇ 10 6 /only), tumor Growing for 28 days.
  • HCT-116+PBMC cells were inoculated intraperitoneally the next day, and administered once every 2 days for 10 consecutive administrations, IL-15 was 2 ⁇ g/head, 3 was 10 ⁇ g/head, and 7 was 20 ⁇ g/head ( IL-15, 3 and 7 are equimolar, both 0.15 nmol).
  • the tumor volume was weighed every 2-3 days from day 12, weighed, and data were recorded. On the 28th day, he was killed and taken a tumor.
  • V 1/2 ⁇ L length ⁇ L short 2
  • Tumor inhibition rate (%) (V T - V 0 ) / V T (%)
  • V 0 and V T are the tumor volume at the beginning of the experiment and at the end of the experiment, respectively.
  • IL-15, dimeric protein 3 and dimeric protein 7 were administered in the same manner for 20 days after the cells were inoculated, and administered once every 2 days.
  • the inhibitory effect of IL-15 protein on the growth of HCT-116+PBMC tumors is shown in Table 4 and Figure 6. No tumors were found in each group in the first week. From day 12 (D12), tumors appeared in each group, and the tumor volume of IL-15, dimeric protein 3, and dimeric protein 7 group was smaller than that of PBMC group.
  • D28 day 28
  • IL-15 and dimeric protein 3 groups significantly inhibited the growth of HCT-16 cell xenografts, with inhibition rates of 42% and 41%, respectively; and dimeric protein 7 group inhibition rate of 27%. However, it was not significantly different from the PBMC group. There was no death of the mice during the administration, and there was no significant decrease in body weight during the administration of the mice in each group, suggesting that there is no obvious side effect at the current dose.
  • the tumor weight of each group was stripped. As shown in Fig. 7, the tumor weight of the dimeric protein 3 group was significantly lower than that of the PBMC group; the tumor weight of the IL-15 group was lower than that of the PBMC group, but there was no Significant; the tumor weight of the dimeric protein 7 group was comparable to that of the PBMC group.
  • Mo7e human megakaryocyte leukemia cell line
  • IL-15 was purchased from Novoprotein, Cat. No. C016, IL-15 analog (dimeric protein 11-17) was prepared from the inside;
  • CCK-8 Cell Counting Kit-8 (CCK-8), purchased from WST, item number EX660;
  • GM-CSF was purchased from NOVO Protein under the product number CC79.
  • Mo7e was cultured in a modified RPMI-1640 medium (containing 2.05 mM L-glutamine, 10% FBS and 15 ng/ml GM-CSF) in a 37 ° C (5% CO 2 ) cell culture incubator;
  • IL-15 and its analogs were diluted four times with PBS, and cultured in 96-well plates for 2 hours, then added to the cell culture system, 10 ⁇ l/well, each receiving The test substance concentration was repeated three times and a blank control well (only PBS was added);
  • the cell culture plate is further cultured for 3 days in an incubator
  • the molecular dimeric protein 11-17 to be tested was significantly superior to the IL15 control molecule in the Mo7e proliferation assay.
  • mice 32 C57BL/6 mice (SPF, Shanghai Xipuer Bikai Experimental Animal Co., Ltd.), through the tail vein injection of 1.5 ⁇ 10 5 B16F10 cells (Chinese Academy of Sciences Shanghai Institute of Life Sciences Cell Resource Center, item number TCM36), points There are 4 groups of 8 each.
  • PBS, 2 ⁇ g of IL-15, 4.2 ⁇ g, and 12.5 ⁇ g of dimeric protein 17 were intraperitoneally injected, and weighed once every 2-3 days, and one of each group died on the 14th day, and lung metastasis was observed. All mice were sacrificed on the 16th day, the lungs were removed, weighed, black tumor spots in the lungs were observed, photographed, fixed in methanol, and the number of black tumor spots was counted.
  • FIG. 8 shows the number of lung metastasis tumor spots in each group of test mice, compared with the PBS group, * indicates p ⁇ 0.05, ** indicates p ⁇ 0.01.
  • the relative weight of the lungs in the PBS group was higher than that in the dimeric protein 17 administration group, as shown in FIG.
  • mice in the dimer protein 17-12.5 ug dose group showed a slight decrease in body weight on the 5th day after administration, and then gradually recovered, as shown in FIG.
  • dimeric protein 17 is capable of inhibiting lung metastasis of B16F10 cells in mice and has a dose effect.

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Abstract

公开了一种IL-15异源二聚体蛋白及其用途。所述IL-15异源二聚体蛋白包含蛋白(Ⅰ)和蛋白(Ⅱ)，其中蛋白(Ⅰ)由IL-15或其变体和第一Fc变体重组而成；蛋白(Ⅱ)是第二Fc变体，或由IL-15Rα或其变体和第二Fc变体重组而成；第一Fc变体和第二Fc变体间以Knob-into-Hole形式连接。所述IL-15异源二聚体蛋白具有明显的抑瘤活性，比单一IL-15分子具有更好的生物活性及延长的体内半衰期。

Description

IL-15异源二聚体蛋白及其用途 技术领域
本发明涉及一种IL-15异源二聚体蛋白及其用途，进一步涉及一种IL-15/IL-15Rα异源二聚体蛋白复合物，以及其作为治疗剂特别是作为癌症和自身免疫疾病治疗剂的用途。
背景技术
白细胞介素15(IL-15)是Grabstein等人于1994年发现的一种约为12-14kD的细胞因子，可在机体正常的免疫应答中发挥作用，如促进T细胞、B细胞、自然杀伤(NK)细胞的增殖。
IL-15属于四个小α螺旋束细胞因子家族(Small four α-helix bundle family of cytokines)中的成员。IL-15需通过与其受体结合发挥生物学活性。IL-15受体由三个受体亚基组成：IL-15受体α(IL-15Rα)、IL-2受体β(IL-2Rβ，也称IL-15Rβ或CD122)和γc(也称CD132)。IL-15Rα内含一个Sushi结构域，能与IL-15结合，并且是使结合后的IL-15发挥生物学功能所必需的。
近年发现，IL-15与其受体IL-15Rα形成复合物后，可显著增强IL-15的生物学活性。研究显示，IL-15和其可溶性受体IL-15Rα所形成的复合物在刺激记忆性CD8+T淋巴细胞和NT/NKT细胞增殖作用明显地优于IL-15单独作用。IL-15/IL-15Rα复合物刺激记忆性CD8+T细胞增殖和维持其存活的能力较单独的IL-15强10倍以上，其机理可能和顺式递呈相关。
由于IL-15在肿瘤免疫治疗领域的良好预期，NIH最先进行了IL-15治疗肿瘤方面的研究，并尝试将其推入临床一期研究。但是IL-15分子量小、体内半衰期段，重复给药剂量不好控制，且容易造成全身性免疫副反应等。所以迫切需要一种提高IL-15体内半衰期、促进或者增强其体内生物学活性发挥的途径。国内外有不少公司或研究机构在从事IL-15免疫治疗方面的相关研究，比如CN100334112C(上海海欣生物技术有限公司)专利所涉及的IL-15-hIgG4Fc同源二聚体蛋白用于抗微生物感染的治疗；如CN1942481A(瑞士豪夫迈-罗氏公司)专利所涉及的IL-15-Fc融合表达系统及其用途；如CN101360827B(法国国立医学与健康研究所)专利所涉及的IL-15Ra(sushi+结构域)-IL-15融合蛋白及其在肿瘤治疗方面的应用。本申请的异源二聚体分子通过增加分子内部相互作用，具有更好的稳定性、延长的体内半衰期及提高的生物学活性。通过本领域内熟知的方法手段可以在本申请的分子设计基础上通过融合或者插入功能多肽的方式产生靶向性免疫细胞因子以及靶向性免疫细胞因子在肿瘤及自身免疫疾病治疗领域的应用。
发明内容
本发明通过基因工程的方法设计并制备具有体内半衰期延长的，体外活性提高的并有明显抑瘤活性的蛋白分子。
本发明提供一种IL-15异源二聚体蛋白，其包括：
蛋白(I)和蛋白(Π)；
其中蛋白(I)由IL-15或其变体和第一Fc变体重组而成；
蛋白(Π)是第二Fc变体，或者由IL-15Rα或其变体和第二Fc变体重组而成；
蛋白(I)和蛋白(Π)通过第一Fc变体和第二Fc变体之间的相互作用形成稳定的异源二聚体蛋白。
以上所述的”重组而成”指通过基因工程的方法由不同蛋白间重组后表达得到。
在本发明一个优选的实施方案中，第一Fc变体和第二Fc变体连接在蛋白IL-15和IL-15Rα的C端。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的IL-15序列为SEQ ID NO：1。
在本发明一个优选的实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的蛋白(Π)是第二Fc变体。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的蛋白(Π)由IL-15Rα或其变体和第二Fc变体重组而成。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的IL-15Rα变体为IL-15Rα胞外域部分或其功能性片段，所述的功能性片段优选为IL-15Rα胞外域部分65-120个氨基酸的缩短形式，更优选65-102个氨基酸的缩短形式。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的IL-15Rα变体的序列选自SEQ ID NO：2-7。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的IL-15Rα变体序列选自SEQ ID NO：3-7。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述的第一Fc变体和第二Fc变体分别选自Knob修饰的Fc和Hole修饰的Fc，或所述的第一Fc变体和第二Fc变体分别选自Hole修饰的Fc和Knob修饰的Fc。修饰后的第一Fc变体和第二Fc变体通过“Knob/Hole”的作用方式有助于形成异源二聚体蛋白。当第一Fc变体是Knob修饰的Fc时，第二Fc变体是Hole修饰的Fc；或者当第二Fc变体是Knob修饰的Fc时，第一Fc变体是Hole修饰的Fc。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述第一Fc变体的序列选自SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29，和其中所述第二Fc变体的序列选自SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29。蛋白(I)和蛋白(Π)通过如SEQ ID NO： 26和SEQ ID NO：27或SEQ ID NO：28和SEQ ID NO：29所示的Fc变体以“Knob/Hole”的作用方式形成异源二聚体。如当第一Fc变体序列为SEQ ID NO：26时，第二Fc变体序列为SEQ ID NO：27；或者当第二Fc变体序列为SEQ ID NO：26时，第一Fc变体序列为SEQ ID NO：27。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述蛋白(I)的序列选自SEQ ID NO：14-17，优选是SEQ ID NO：14。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述蛋白(Π)的序列选自SEQ ID NO：18-25、34-37，优选选自SEQ ID NO：23、34-37，更优选选自SEQ ID NO：34-37。
在本发明一个实施方案中，提供一种IL-15异源二聚体蛋白，其中所述蛋白(I)的序列选自SEQ ID NO：30-31；所述蛋白(Π)的序列选自SEQ ID NO：32-33。
本发明的IL-15异源二聚体蛋白，选自如下的二聚体蛋白3-17，其中所述的二聚体蛋白3-17分别由相对应的蛋白(I)和蛋白(Π)重组而成：
Figure PCTCN2014094947-appb-000001
本发明还涉及一种核酸，其编码如上所述的IL-15异源二聚体蛋白。
本发明还涉及一种DNA载体，其包括如上所述的核酸。
本发明还涉及一种宿主细胞，其转染有本发明所述的DNA载体。
本发明还涉及一种制备如上所述的IL-15异源二聚体蛋白的方法，所述方法包括：在足以表达如上所述的IL-15异源二聚体蛋白的条件下，培养本发明所述的宿主细胞；表达并纯化所述的IL-15异源二聚体蛋白。
本发明还涉及一种药物组合物，其含有本发明所述的IL-15的异源二聚体蛋白和可药用的赋形剂、稀释或载体。
本发明还涉及一种靶向性蛋白分子，其含有根据本发明所述的IL-15异源二聚体蛋白结构。
本发明还涉及一种用于刺激或抑制哺乳动物免疫应答的方法，包括：向所述 哺乳动物给予有效量的根据本发明所述的IL-15异源二聚体蛋白，或根据本发明所述的药物组合物，或根据本发明所述的靶向性蛋白分子。
本发明还涉及根据本发明所述的IL-15异源二聚体蛋白、或根据本发明所述的药物组合物，或根据本发明所述的靶向性蛋白分子，在制备用于治疗IL-15介导的疾病或病症的药物中的用途；其中所述的疾病为传染病、癌症、血液病和自身免疫性疾病。所述的癌症选自黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤、肝癌、肺癌、胃癌、乳腺癌；所述的传染病选自天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；所述的血液病选自贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病选自多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎。其中所述的药物为根据本发明所述的IL-15异源二聚体蛋白、或本发明所述的药物组合物与小分子抑制剂或抗体类药物的联合使用；所述的小分子抑制剂优选烷化剂；所述的抗体类药物优选为单克隆抗体药物，更优选为抗CD20、PD1、PDL1、Her2抗体。进一步地，根据本发明所述的药物与治疗有效剂量的选自下列的药物联合应用：替莫唑胺、阿霉素、紫杉醇、顺铂、卡铂、达卡巴嗪、拓扑替康、伊立替康、吉西他滨和贝伐单抗。
本发明还涉及根据本发明所述的IL-15异源二聚体蛋白、或根据本发明所述的药物组合物，或根据本发明所述的靶向性蛋白分子，在细胞免疫治疗中的用途，尤其是在DC、CIK、DC-CIK、ECIK、NK、CAS-T、BiAb-T、TCR-T、CAR-T肿瘤免疫细胞治疗中的用途。
肿瘤免疫治疗是肿瘤治疗领域一个热点，是继手术、化疗和放疗之后的第四种肿瘤治疗模式。肿瘤免疫学治疗的目的是激发或调动机体的免疫系统，增强肿瘤微环境抗肿瘤免疫力，从而控制和杀伤肿瘤细胞。它可能是最有效的也是最安全的治疗肿瘤的途径。
肿瘤的免疫逃逸机制，是利用肿瘤自身对免疫系统的抑制作用维持或促进肿瘤的生长。肿瘤免疫治疗是为了最大限度的提高患者自身对肿瘤的免疫系统反应，它不但要在体内激活原有的免疫系统反应，更要维持免疫系统反应的持续时间和反应强度，这才是免疫治疗肿瘤的关键。
细胞因子免疫疗法是随着高纯度或重组细胞因子的生产而发展起来的。其原理是利用某些细胞因子注射体内后可调节、增强一种或多种免疫细胞的功能，发挥更强的抗肿瘤免疫作用。
本发明还涉及一种治疗或预防疾病的方法，在所述疾病中，细胞表达疾病相关抗原，所述方法包括：向患者施予如本发明所述的IL-15异源二聚体蛋白、或如本发明所述的药物组合物，或如本发明所述的靶向性蛋白分子；在表达疾病相关抗原的细胞与表达IL-15Rα的免疫细胞之间形成足以活化所述免疫细胞的特异性结合复合物；以及通过所述免疫细胞杀死所述表达疾病相关抗原的细胞。其中所 述表达疾病相关抗原的细胞优选为肿瘤细胞或病毒感染细胞。其中所述的免疫细胞优选为T-细胞、LAK细胞或NK细胞。其中所述的疾病为传染病、癌症、血液病和自身免疫性疾病。所述的癌症选自黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、实体瘤；所述的传染病选自天花病毒感染、HIV感染、细菌感染、真菌感染；所述的血液病选自贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；所述的自身免疫性疾病选自多发性硬化症、银屑病、风湿性关节炎、炎性疾病、胃炎、黏膜炎。
本发明还涉及一种治疗或预防疾病的方法，包括向患者施予如本发明所述的IL-15异源二聚体蛋白、或如本发明所述的药物组合物，或如本发明所述的靶向性蛋白分子，以及联合施用其它药物，如小分子抑制剂或抗体类药物；所述的小分子抑制剂优选烷化剂；所述的抗体类药物优选单克隆抗体药物，更优选抗CD20、PD1、PDL1、Her2抗体。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
一、术语
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的“异源二聚体蛋白”是指两个不同的单体蛋白质结合而成的蛋白。在本发明中，两个不同的单体蛋白质各自含有Fc片段或Fc变体片段，并通过Fc片段或Fc变体片段相互作用形成异源二聚体蛋白。
本发明中第一Fc变体和第二Fc变体之间的“相互作用”指的是Fc变体间作用。“Fc变体”指通过在Fc上合适的位点处存在一个或者多个氨基酸替换、插入或缺失突变引起Fc结构或功能的变化。“Fc变体间作用”指的是经突变设计的Fc变体之间，可以形成空间填充效应、静电导引、氢键作用、疏水作用等。Fc变体间相互作用有助于形成稳定的异源二聚体蛋白。优选的突变设计为“Knob-into-Hole”形式的突变设计。
本发明中组成异源二聚体蛋白的“单体蛋白质”(即蛋白(I)、蛋白(Π))可以是融合蛋白或非融合蛋白。
本发明所述的“融合蛋白”是指，通过用基因重组方法、化学方法或其它适当方法将两个或多个基因的编码区连接，在同一调控序列控制下表达基因重组所得的蛋白质产物。在本发明的一些实施方案中，蛋白(I)为IL-15或其变体与Fc变体基因重组表达得到的融合蛋白；蛋白(Π)可为IL-15Rα与Fc变体基因重组表达得到的融合蛋白。本发明的融合蛋白中，两个或多个基因的编码区之间可由编码肽接头的序列于一个或数个位置融合。肽接头也可用于构建本发明的融合蛋白。
本发明所述的“IL-15”或“IL-15肽段”可以是任何IL-15(白介素15)或其突变体，如人IL-15或非人哺乳动物IL-15或非哺乳动物IL-15。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。；优选人的白介素15成熟分子，见数据库UniProtKB，登录号P40933，49-162aa。术语“IL-15变体”指通过一个或多个氨基酸替换、增加或者缺失突变获得的对IL-15与其受体间亲合力提高或者降低，或其刺激T细胞或者NK细胞活性增加或者降低的突变体分子。
本发明中所述的“IL-15Rα”可以是任何物种的IL-15Ra或者其功能性片段，如人IL-15Ra或非人哺乳动物IL-15Ra或非哺乳动物IL-15Ra。示例性非人哺乳动物如猪、兔、猴、猩猩、鼠等，非哺乳动物如鸡等。优选人的IL-15Ra，；更优选人的白介素15受体α胞外域片段，简称IL-15RαECD(SEQ ID NO：2)，见数据库UniProtKB，登录号Q13261，31-205aa。术语“IL-15Rα变体”指在IL-15Ra上通过一个或者多个氨基酸缺失、插入或替换突变形成的具有与其配体分子如IL15结合能力的功能性突变体，优选人的IL15Ra分子，更优选人的IL-15Rα胞外域片段的缩短形式，即从胞外域片段C端开始通过一个或多个氨基酸缺失突变所得的具有人白介素15受体α活性的分子，优选保留65-120个氨基酸的缺失突变形式，更优选保留65-102个氨基酸的缺失突变缩短形式，比如IL-15Rα-sushi(77)(SEQ ID NO：3)、IL-15Rα-sushi(65)(SEQ ID NO：4)。
术语“免疫球蛋白Fc区”指的是免疫球蛋白链恒定区，特别是免疫球蛋白重链恒定区的羧基端或其中的一部分，无抗原结合活性，是抗体分子与效应分子和细胞相互作用的部位。例如，免疫球蛋白Fc区可包括重链CH1、CH2、CH3、CH4的两个或更多结构域与免疫球蛋白铰链区的组合。Fc可以来源于不同的种属，优选人的免疫球蛋白。根据重链恒定区的氨基酸序列，免疫球蛋白可以分为不同的种类，主要有5类免疫球蛋白：IgA、IgD、IgE、IgG和IgM。其中一些还可进一步分成亚类(同种型)，如IgG-1、IgG-2、IgG-3、IgG-4；IgA-1和IgA-2。
“Fc区”优选包括至少一个免疫球蛋白绞链区，以及IgG的CH2和CH3区。更优选包括IgG1的一个CH2结构域，一个CH3结构域和一个免疫球蛋白绞链区，铰链区起始氨基酸位置可以变动。
Fc变体的突变设计技术在本领域内已经较为广泛的应用于制备双特异性抗体或者异源二聚的Fc融合蛋白形式。代表性的有Cater等人(Protein Engineering vol.9no.7pp.617-621,1996)提出的“Knob-into-Hole”形式；Amgen公司技术人员利用静电导引(Electrostatic Steering)形成含Fc的异源二聚体形式(US 20100286374A1)；Jonathan H.Davis等人(Protein Engineering,Design&Selection pp.1–8,2010)提出的通过IgG/IgA链交换形成的异源二聚体形式(SEEDbodies)；Genmab公司DuoBody(Science,2007.317(5844))平台技术形成的双特异性分子；Xencor公司的技术人员综合结构计算及Fc氨基酸突变，综合不同作用方式形成异源二聚体蛋白形式(mAbs 3:6,546-557；November/December 2011)；苏州康宁杰瑞公司的 基于电荷网络的Fc改造方法(CN201110459100.7)得到异源二聚体蛋白形式；以及其它基于Fc氨基酸变化或者功能改造手段，达到形成异源二聚体功能蛋白的基因工程方法。本发明所述的Fc变体片段上的Knob/Hole结构指两条Fc片段各自突变，突变后可以通过“Knob-into-Hole”形式进行结合。优选用Cater等人的“knob-into-hole”模型在Fc区上进行位点突变的改造，以使得到的第一Fc变体和第二Fc变体能以“knob-into-hole”的形式结合在一起形成异源二聚体。从特定的免疫球蛋白类别和亚类中选择特定的免疫球蛋白Fc区在本领域技术人员所掌握的范围之内。优选人类抗体IgG1、IgG2、IgG3、IgG4的Fc区，更优选人抗体IgG1的Fc区。随机任选第一Fc变体或第二Fc变体中一个做knob的突变，另一个做hole的突变。在实施例中，所述的第一Fc变体做knob的突变，如序列SEQ ID NO26；所述的第二Fc变体做hole的突变，如序列SEQ ID NO 27。
术语“连接肽(Linker)”在本发明中用于连接IL-15或IL-15Rα与Fc变体，以保证蛋白的正确折叠和稳定性的肽。本发明的“连接肽”优选为(GGGGS)n，其中n可以为0、1、2、3、4、5或者更多，优选n为2-4。如果连接肽序列太短，可能影响两蛋白高级结构的折叠，从而相互干扰；如果连接肽序列太长，又涉及免疫原性的问题，因为连接肽序列本身就是新的抗原。
本发明所述的“异源二聚体蛋白”优选为基因共表达的产物。如在原核细胞在肠杆菌中共表达；或在真核细胞，如293、CHO中共表达。
本发明中所述的“共表达”(coexpression)指在一个细胞中多个基因一起表达，同时出现它们的产物。这些基因可以是同时存在而分别或共同地受控表达。在本发明中，优选在一个真核细胞中共表达两种基因。共表达得到的基因表达产物有利于高效、简单地形成复合物；在本发明中，有利于形成异源二聚体蛋白。
本发明所述的免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。免疫球蛋白重链恒定区的氨基酸组成和排列顺序不同，故其抗原性也不同。据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ或λ链。
靶向性蛋白分子，指一类蛋白分子，其中含有某个片段或者区域能够和其他蛋白分子相互作用，通过在免疫细胞因子如IL5、IL2等或者含有这类细胞因子的独特分子设计比如本申请的分子设计基础上引入靶向性肽段如抗体片段、ScFv或某类细胞表面分子的结合肽等。例如抗体与抗原相互作用，或者配体与受体相互作用，使得分子进入体内后能够优先通过靶向作用富集于特定的组织、器官或身体部位发挥其生物功能。通过如在本申请分子的游离末端融合某类多肽形成一个新的分子，这种方式在本领域内可以合理的进行衍生设计产生一系列分子。
“给予”和“处理”，当应用于动物、人、受试者、细胞、组织、器官或生物流 体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”，当应用于人、动物或受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。“处理”在应用于人、动物或受试者、或者细胞、组织或器官时，包括IL-15激动剂或IL-15拮抗剂与人或动物、受试者、细胞、组织、生理区室或生理流体的接触。“细胞的处理”还包括IL-15激动剂或IL-15拮抗剂例如在流体相或胶体相中接触IL-15受体的情况，而且包括激动剂或拮抗剂不接触细胞或受体的情况。
“治疗”意指给予患者内用或外用治疗剂，诸如包含本发明的IL-15异源二聚体蛋白的组合物。所述患者具有一种或多种疾病或症状。已知所述治疗剂对这些疾病或症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病或症状的量给予治疗剂，无论是通过诱导这类疾病或症状退化还是抑制这类疾病或症状发展到任何临床可测量的程度。有效缓解任何具体疾病或症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。
“免疫病症”或“免疫障碍”包括例如病理性炎症、炎性病症和自身免疫性疾病症或疾病。“免疫病症”还指感染、持续感染和增生性病症，例如癌症、肿瘤和血管发生。“癌性病症”包括例如癌症、癌细胞、肿瘤、血管发生和癌变前病症，例如发育异常。
本文使用的“聚合酶链式反应”或“PCR”是指例如美国专利号4,683,195中所述的扩增程序或技术。一般来说，需要获得来自目标区域末端或之外的序列信息，使得可以设计寡核苷酸引物。这些引物的序列与待扩增模板的对应链相同或相似。
“任选”或“任选地”意味着随后所描述地事件或环境可以但不必发生，该说明包括该事件或环境发生或不发生地场合。例如，“任选包含1-3个抗体重链可变区”意味着抗体重链可变区可以但不必须存在；存在时，可以是1个，2个或3个。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其它化学组分的混合物，以及其它组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。
本发明中所述的用重组DNA转化宿主细胞的步骤可用本领域技术人员熟知的常规技术进行。获得的转化子可以用常规方法培养，以及表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。宿主细胞在适于宿主细胞生长的条件下进行培养。
附图说明
图1.给药时间与血清中样本的摩尔浓度的关系。
图2.测试例3.肺转移模型中，给药的小鼠肺部转移肿瘤块数量。图中*表示：p<0.05,vsPBS；**表示：p<0.01,vsPBS；##表示：p<0.01,vs IL-15。
图3.测试例3.肺转移模型中，给药的小鼠相对肺重量(肺重量/体重)，图中**表示：p<0.01,vs PBS；#表示：p<0.05,vs IL-15。
图4.不同给药蛋白对HCT-116裸小鼠移植瘤的疗效。
图5.不同给药蛋白对HCT-116裸小鼠移植瘤的疗效，第27天的肿瘤重量。图中*表示：p<0.05，vs blank(空白对照)。
图6.不同给药蛋白对HCT-116+PBMC SCID鼠移植瘤的疗效。
图7.不同给药蛋白对HCT-116+PBMC SCID鼠移植瘤的疗效，第28天的肿瘤重量。图中*表示：p<0.05，vs PBMC。
图8.小鼠给药二聚体蛋白17、阳性对照(IL-15)和阴性对照(PBS)后小鼠的肺部转移肿瘤斑点数量比较，图中*：p<0.05，**：p<0.01，vs PBS。
图9.向小鼠给药二聚体蛋白17、阳性对照(IL-15)和阴性对照(PBS)后小鼠的相对肺重量(肺重量/体重)比较。
图10.向小鼠给药二聚体蛋白17、阳性对照(IL-15)和阴性对照(PBS)后小鼠的小鼠体重比较。
具体实施方式
以下结合实施例用于进一步描述本发明，但这些实施例并非限制本发明的范围。
本发明实施例或测试例中未注明具体条件的实验方法，通常按照常规条件，或按照原料或商品制造厂商所建议的条件。参见Sambrook等，分子克隆，实验室手册，冷泉港实验室；当代分子生物学方法，Ausubel等著，Greene出版协会，Wiley Interscience，NY。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.IL-15异源二聚体蛋白的获得
本发明提供的异源二聚体蛋白由蛋白(I)和蛋白(Π)结合形成，所述的蛋白(I)是由IL-15或其变体和第一Fc变体重组而成的融合蛋白；所述的蛋白(Π)，可以是第二Fc变体，或者是由IL-15RαECD或其变体和第二Fc变体重组而成的融合蛋白；所述的结合优选第一Fc变体与第二Fc变体以Knob/Hole形式结合。
本发明实施例中所用的IL-15指的是人白介素15成熟分子(SEQ ID NO：1)或其变体。本发明实施例中所用的IL-15RαECD指人的白介素15受体α胞外域片段(SEQ ID NO：2)；其变体优选其缩短形式，如IL-15Rα-sushi(77)(SEQ ID  NO：3)、IL-15Rα-sushi(65)(SEQ ID NO：4)。本发明实施例中所用的Fc片段部分可以为人类抗体IgG1、IgG2、IgG3、IgG4的Fc片段，优选人IgG1的Fc片段。本发明中第一Fc变体或第二Fc变体，优选进行Knob形式的突变(SEQ ID NO：26)，或Hole形式的突变(SEQ ID NO：27)。本发明的异源二聚体蛋白中，蛋白(I)和蛋白(Π)之间通过第一Fc变体与第二Fc变体之间的Knob/Hole结构形成二聚体，优选通过第一Fc变体与第二Fc变体之间“Knob-into-Hole”作用方式形成异二聚体。仅含单个IL-15效应分子的二聚体，也可以通过IL-15与第一Fc变体融合，且所述第一Fc变体与相对应第二Fc变体以Knob-into-Hole的作用方式形成，如本发明的异源二聚体蛋白3。
本发明将IL-15或其变体通过一个连接肽与第一Fc片段或第一Fc变体融合形成融合蛋白，以及本发明将IL-15RαECD或其变体通过一个连接肽与第二Fc片段或第二Fc变体融合形成融合蛋白，其中各蛋白组分的连接顺序不限；连接肽可以是领域内常用的软性连接肽，优选为(GGGGS)n，其中n选自1-10，优选选自1-5，最优选为2。
相关的蛋白序列如下：
IL-15(蛋白序列1):(人白介素15的氨基酸序列，同时也是对照品IL-15序列)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO：1。
IL-15RαECD(蛋白序列2):(人白介素15受体alpha的胞外域氨基酸序列，同时也用来和Fc融合形成分子H中的IL-15Ra所指部分)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT
SEQ ID NO：2。
IL-15Rα-sushi(77)(蛋白序列3):(人白介素15受体alpha胞外域中能够保持90％以上结合活性的一个结构域，称为sushi结构域加一小段连接肽，属于缩短形式的IL-15Rα)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO：3。
IL-15Rα-sushi(65)(蛋白序列4)：(人白介素15受体alpha胞外域中能够保持90％以上结合活性的一个结构域，称为sushi结构域属于缩短形式的IL-15Ra)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SEQ ID NO：4。
IL15Ra-sushi(73)(蛋白序列5)：
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAH  WTTPSLKCIRDPALVHQR SEQ ID NO：5
IL15Ra-sushi(86)(蛋白序列6)：
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVT SEQ ID NO：6
IL15Ra-sushi(102)(蛋白序列7)：
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAAS
SEQ ID NO：7
IL-15-Fc(蛋白序列8):(人白介素15分子通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，表达出来为双价的同源二聚体，其中IL-15分子处在蛋白的N端)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：8。
Fc-IL-15(蛋白序列9):(人白介素15分子通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，表达出来为双价的同源二聚体，其中IL-15分子处在蛋白的C端)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO：9。
IL-15RαECD-Fc(蛋白序列10):(人白介素15受体alpha胞外域通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，表达出来为双价的同源二聚体，其中IL-15Rα-ECD分子处在蛋白的N端)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO： 10。
Fc-IL-15RαECD(蛋白序列11):(人白介素15受体alpha胞外域通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，表达出来为双价的同源二聚体，其中IL-15RαECD分子处在蛋白的C端)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT SEQ ID NO：11。
IL-15Rα-sushi(77)-Fc(蛋白序列12):(人白介素15受体alpha胞外域中的sushi结构域加一段连接肽组成的sushi(77)片段通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，sushi(77)片段位于N端)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：12。
Fc-IL-15Rα-sushi(77)(蛋白序列13):(人白介素15受体alpha胞外域中的sushi结构域加一段连接肽组成的sushi+片段通过一个连接肽与人IgG1-Fc序列连接形成的融合蛋白，sushi+片段位于C端)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO：13。
IL-15-Fc-Knob(蛋白序列14):(上述序列8中的Fc部分进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:14。
IL-15-Fc-Hole(蛋白序列15):(上述序列8中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：15。
Fc-Knob-IL-15(蛋白序列16):(上述序列9中的Fc部分进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO：16。
Fc-Hole-IL-15(蛋白序列17):(上述序列9中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SEQ ID NO：17。
IL-15RαECD-Fc-Knob(蛋白序列18):(上述序列10中的Fc部分进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:18。
IL-15RαECD-Fc-Hole(蛋白序列19):(上述序列10中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTGGGGSGGGGSEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW  YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：19.
Fc-Knob-IL-15RαECD(蛋白序列20):(上述序列11中的Fc部分进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT SEQ ID NO:20。
Fc-Hole-IL-15RαECD(蛋白序列21):(上述序列11中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTT SEQ ID NO:21。
IL-15Rα-sushi(77)-Fc-Knob(蛋白序列22):(蛋白序列12中的Fc部分进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：22。
IL-15Rα-sushi(77)-Fc-Hole(蛋白序列23):(蛋白序列12中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：23。
Fc-Knob-IL-15Rα-sushi(77)(蛋白序列24):(蛋白序列13中的Fc部分 进行了氨基酸突变成Knob形式，可以和另外一个融合分子的Hole形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO：24。
Fc-Hole-IL-15Rα-sushi(77)(蛋白序列25):(蛋白序列13中的Fc部分进行了氨基酸突变成Hole形式，可以和另外一个融合分子的Knob形式进行配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSGGSGGGGSGGGSGGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPP SEQ ID NO：25。
Fc-Knob(蛋白序列26)：(人IgG1-Fc部分的Knob突变形式，可以与IL-15-Fc-Hole/Fc-IL-15-Hole配对)
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：26。
Fc-Hole(蛋白序列27)：(人IgG1-Fc部分的Hole突变形式，可以与IL-15-Fc-Knob/Fc-IL-15-Knob配对
EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：27。
Fc-Knob(M)(蛋白序列28)：另一种方式的Fc突变，可以和Fc-Hole(M)配对形成异源二聚体。
EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：28
Fc-Hole(M)(蛋白序列29)：另一种方式的Fc突变，可以和Fc-Knob(M) 配对形成异源二聚体。
EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：29
Fc-Knob(M)-IL-15(蛋白序列30)(相比Knob突变位点不同，另外一种异源二聚体突变方式)
EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS
SEQ ID NO：30
IL-15-Fc-Knob(M)(蛋白序列31)(相比Knob突变位点不同，另外一种异源二聚体突变方式)
NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSGGGGSEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：31
Fc-Hole(M)-IL-15Rα-sushi(65)(蛋白序列32)(相比Hole突变位点不同，另外一种异源二聚体突变方式)
EPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR
SEQ ID NO：32
IL-15Rα-sushi(65)-Fc-Hole(M)(蛋白序列33)(相比Hole突变位点不 同，另外一种异源二聚体突变方式)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRGGGGSGGGGSGGGGSGGGGSEPKSSDKTHTSPPSPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：33
IL-15Rα-sushi(73)-Fc-Hole(蛋白序列34)：(sushi(73)指长度为73个氨基酸的含sushi结构域的缩短形式的IL15Rα)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：34
IL-15Rα-sushi(65)-Fc-Hole(蛋白序列35)：(sushi(65)指65个氨基酸长度的sushi结构域)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO：35
IL-15Rα-sushi(86)-Fc-Hole(蛋白序列36)：(sushi(86)指长度为86个氨基酸的含sushi结构域的缩短形式的IL-15Rα)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：36
IL-15Rα-sushi(102)-Fc-Hole(蛋白序列37)：(sushi(102)指长度为102个氨基酸的含sushi结构域的缩短形式的IL-15Rα)
ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSGGSGGGGSGGGSGGGGSLQEPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO：37
实施例2相关载体的构建
一、实验材料：
真核表达载体pcDNA3.1(+)(Life technologies，货号V790-20)；
IL-15(DNA序列1)、IL-15RαECD(DNA序列2)及IgG1Fc(DNA序列3)DNA片段：由基因合成公司合成(金唯智，苏州)；
引物DNA片段：由基因合成公司合成(金唯智，苏州)。
二、实验方法：
用常规的PCR方法，进行片段拼接。
1.片段拼接
IL-15-Fc片段：以IL-15，连接肽，Fc的顺序，进行overlap PCR，将三个DNA片段拼接成IL-15-Fc片段(DNA序列4，SEQ ID NO：42)。
IL-15RαECD-Fc片段：以IL-15RαECD，连接肽，Fc的顺序，进行overlap PCR，将三个DNA片段拼接成IL-15RαECD-Fc片段(DNA序列5，SEQ ID NO：43)。
Fc-IL-15片段：以Fc，连接肽，IL-15的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-IL-15片段(DNA序列6，SEQ ID NO：44)。
Fc-IL-15RαECD片段：以Fc，连接肽，IL-15RαECD的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-IL-15RαECD片段(DNA序列7，SEQ ID NO：45)。
IL-15-Fc-Knob片段：以IL-15，连接肽，Fc-Knob的顺序，进行overlap PCR，将三个DNA片段拼接成IL-15-Fc-Knob片段(DNA序列8，SEQ ID NO：46)。
IL-15-Fc-Hole片段：以IL-15，连接肽，Fc-Hole的顺序，进行overlap PCR，将三个DNA片段拼接成IL-15-Fc-Hole片段(DNA序列9，SEQ ID NO：47)。
Fc-Knob-IL-15片段：以Fc-Knob，连接肽，IL-15的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-Knob-IL-15片段(DNA序列10，SEQ ID NO：48)。
Fc-Hole-IL-15片段：以Fc-Hole，连接肽，IL-15的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-Hole-IL-15片段(DNA序列11，SEQ ID NO：49)。
IL-15RαECD-Fc-Knob片段：以IL-15RαECD，连接肽，Fc-Knob的顺序，进 行overlap PCR，将三个DNA片段拼接成IL-15RαECD-Fc-Knob片段(DNA序列12，SEQ ID NO：50)。
IL-15RαECD-Fc-Hole片段：以IL-15RαECD，连接肽，Fc-Hole的顺序，进行overlap PCR，将三个DNA片段拼接成IL-15RαECD-Fc-Hole片段(DNA序列13，SEQ ID NO：51)。
Fc-Knob-IL-15RαECD片段：以Fc-Knob，连接肽，IL-15RαECD的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-Knob-IL-15RαECD片段(DNA序列14，SEQ ID NO：52)。
Fc-Hole-IL-15RαECD片段：以Fc-Hole，连接肽，IL-15RαECD的顺序，进行overlap PCR，将三个DNA片段拼接成Fc-Hole-IL-15RαECD片段(DNA序列15，SEQ ID NO：53)。
Fc-Knob片段，DNA序列16，SEQ ID NO：54。
Fc-Hole片段，DNA序列17，SEQ ID NO：55。
Fc-Knob(M)-IL-15，DNA序列18，编码蛋白序列30前体的核苷酸序列，SEQ ID NO：56。
IL-15-Fc-Knob(M)，DNA序列19，编码蛋白序列31前体的核苷酸序列，SEQ ID NO：57。
Fc-Hole(M)-IL-15Rα-sushi(65)，DNA序列20，编码蛋白序列32前体的核苷酸序列，SEQ ID NO：58。
IL15Rα-sushi(65)-Fc-Hole(M)，DNA序列21，编码蛋白序列33前体的核苷酸序列，SEQ ID NO：59。
IL-15Rα-sushi(73)-Fc-Hole，DNA序列22，编码蛋白序列34前体的核苷酸序列，SEQ ID NO：60。
IL-15Rα-sushi(65)-Fc-Hole，DNA序列23，编码蛋白序列35前体的核苷酸序列，SEQ ID NO：61。
IL-15Rα-sushi(86)-Fc-Hole，DNA序列24，编码蛋白序列36前体的核苷酸序列，SEQ ID NO：62。
IL-15Rα-sushi(102)-Fc-Hole，DNA序列25，编码蛋白序列37前体的核苷酸序列，SEQ ID NO：63。
2.引入酶切位点及信号肽序列：
利用PCR方法在基因片段的5’端引入限制性内切酶KpnI位点，Kozak序列，信号肽序列。KpnI位点至基因片段之间的序列为(SEQ ID NO：38)：
ggtaccttgtgcccgggcgccacc
Figure PCTCN2014094947-appb-000002
(下划线为KpnI酶切位点，斜线部分为信号肽序列)；在这三个片段的3’端分别引入终止密码子TGA和NotI限制性内切酶位点。
3.构建表达载体
利用KpnI和NotI限制酶位点将上述基因片段分别插入载体pcDNA3.1(+)，构建成表达载体如pcDNA3.1-IL-15-Fc，pcDNA3.1-IL-15RαECD-Fc，pcDNA3.1-Fc，pcDNA3.1-Fc-IL-15，pcDNA3.1-Fc-IL-15RαECD等，得到相应的表达质粒。
4.基因定点突变
定点突变过程采用KOD试剂盒(TOYOBO Cat.KOD-201)25μL体系：2.5μL10×KOD buffer，2.5μL 2mM dNTPs，1μL引物1(10μM)，1μL引物2(10μM)，0.5μL KOD plus，1μL 25mM MgSO4，16μL ddH2O。合成程序为94℃2分钟，94℃30秒钟，55℃30秒钟，68℃11分钟，扩增25循环后，再68℃11分钟以结束PCR扩增程序。在PCR产物中直接加入DpnI(NEB Cat.R0176L)1μL消化5小时后，用DH5a感受态细胞转化，挑单克隆测序，得到目的质粒。本发明实施例涉及的蛋白3由含DNA序列8(SEQ ID NO：46)和DNA序列17(SEQ ID NO：55)的表达载体表达所得；蛋白7由含DNA序列8(SEQ ID NO：46)和DNA序列13(SEQ ID NO：51)的表达载体表达所得。其他实施例的表达由组成蛋白的DNA序列的表达载体共表达所得。
三、构建表达质粒的核苷酸序列
以下序列为构建载体所用的DNA序列，下划单横线部分为信号肽DNA序列，下划虚线部分为肽接头DNA序列，下划双横线部分为Fc进行Knob/Hole突变的DNA序列。
DNA序列1：(IL-15,人白介素15的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct SEQ ID NO：39。
DNA序列2：(IL-15RαECD,人白介素15受体alpha的胞外域核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcag acaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact SEQ ID NO：40。
DNA序列3：(Fc，人IgG的Fc核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
SEQ ID NO：41。
DNA序列4：(IL-15-Fc，编码蛋白序列5前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttc
Figure PCTCN2014094947-appb-000003
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTG TTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：42。
DNA序列5：(IL-15RαECD-Fc，编码蛋白序列7前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact
Figure PCTCN2014094947-appb-000004
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：43。
DNA序列6：(Fc-IL-15，编码蛋白序列6前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGG AGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
Figure PCTCN2014094947-appb-000005
Figure PCTCN2014094947-appb-000006
aactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct SEQ ID NO：44。
DNA序列7：(Fc-IL-15RαECD，编码蛋白序列8前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
Figure PCTCN2014094947-appb-000007
Figure PCTCN2014094947-appb-000008
atcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact SEQ ID NO：45。
DNA序列8：(IL-15-Fc-Knob，编码蛋白序列11前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaa tgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct
Figure PCTCN2014094947-appb-000009
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCA
Figure PCTCN2014094947-appb-000010
TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：46。
DNA序列9：(IL-15-Fc-Hole，编码蛋白序列12前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct
Figure PCTCN2014094947-appb-000011
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTC
Figure PCTCN2014094947-appb-000012
AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：47。
DNA序列10：(Fc-Knob-IL-15，编码蛋白序列13前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCA
Figure PCTCN2014094947-appb-000013
TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAGaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct
SEQ ID NO：48。
DNA序列11：(Fc-Hole-IL-15，编码蛋白序列14前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTC
Figure PCTCN2014094947-appb-000014
AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAGaactgggtgaatgtaATTagtgatttgaaaaaaattgaagatcttattcaatctatgcatattgatgctactttatatacggaaagtgatgttcacCCGagttgcaaagtaacagcaatgaagtgctttctcttggagttacaagttatttcacttgagtccggcgatgcaagtattcatgatacagtagaaaatctgatcatcTTAgcaaacaacagtttgtcttctaatgggaatgtaacagaatctggatgcaaagaatgtgaggaactggaggaaaaaaatattaaagaatttttgcagagttttgtacatattgtccaaatgttcatcaacacttct
SEQ ID NO：49。
DNA序列12：(IL-15RαECD-Fc-Knob，编码蛋白序列15前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact
Figure PCTCN2014094947-appb-000015
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCA
Figure PCTCN2014094947-appb-000016
TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：50。
DNA序列13：(IL-15RαECD-Fc-Hole，编码蛋白序列16前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact
Figure PCTCN2014094947-appb-000017
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAAC ACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTC
Figure PCTCN2014094947-appb-000018
AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：51。
DNA序列14：(Fc-Knob-IL-15RαECD，编码蛋白序列17前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCA
Figure PCTCN2014094947-appb-000019
TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAGatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact SEQ ID NO：52。
DNA序列15：(Fc-Hole-IL-15RαECD，编码蛋白序列18前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTC
Figure PCTCN2014094947-appb-000020
AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAGatcACCtgccctCCACCTatgtccgtggaacacgcagacatctgggtcaagagctacagcttgtactccCGCgagcgCtacatttgtaactctggtttcaagcgtaaagccggcACCtccagcctgACCgagtgcgtgttgaacAAGGCCACCaatgtcgcccactggacaaccccAagtctcaaatgcattCGCgaccctgccctggttcaccaaCGCccagcgccaccAtccacagtaACCACTgcaggCgtgaccccacagccagagagcctctccccttctggCaaagagccAgcagcttcatctccAagctcaaacaacacagcggccacaacagcagctattgtcccgggctcccagctgatgccttcaaaatcaccttccacaggCaccacagagatCagcagtcatgagtcctcccacggcaccccAtctcagacaacagccaagaactgggaactcacagcatccgcctcccaccagccgccaggtgtgtatccacagggccacagcgacaccact SEQ ID NO：53。
DNA序列16：(Fc-Knob，编码蛋白序列23前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCA
Figure PCTCN2014094947-appb-000021
TGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGTACAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
SEQ ID NO：54。
DNA序列17：(Fc-Hole，编码蛋白序列24前体的核苷酸序列)
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGT TCCCCGGCTCTCGGTGCGAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTC
Figure PCTCN2014094947-appb-000022
AGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
SEQ ID NO：55。
DNA序列18：(Fc-Knob(M)-IL-15，编码蛋白序列30前体的核苷酸序列):
ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCC AGTGCGAGCCCAAATCTAGTGACAAAACTCACACGTCCCCACCGTCCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAAGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
Figure PCTCN2014094947-appb-000023
Figure PCTCN2014094947-appb-000024
AACTGGGTGAATGTAATTAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCGAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGCGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCTTAGCA AACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT SEQ ID NO：56
DNA序列19：(IL-15-Fc-Knob(M)，编码蛋白序列31前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCAACTGGGTGAATGTAATTAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCGAGTTGCAAAGTAACAGCAATGAAGTGCTTTCTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGCGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCTTAGCAAACAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCT
Figure PCTCN2014094947-appb-000025
Figure PCTCN2014094947-appb-000026
GAGCCCAAATCTAGTGACAAAACTCACACGAGCCCACCGAGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAAGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATGCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGTGGTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA SEQ ID NO：57
DNA序列20：(Fc-Hole(M)-IL-15Rα-sushi(65)，编码蛋白序列32前体的核苷酸序列):
ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTCGCGATTCTTAAGGGTGTCC AGTGCGAGCCCAAATCTAGTGACAAAACTCACACGTCCCCACCGTCCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAAGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGA GAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCAtccCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
Figure PCTCN2014094947-appb-000027
Figure PCTCN2014094947-appb-000028
ATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGC
SEQ ID NO：58
DNA序列21：(IL15Rα-sushi(65)-Fc-Hole(M)，编码蛋白序列33前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGC
Figure PCTCN2014094947-appb-000029
Figure PCTCN2014094947-appb-000030
GAGCCCAAATCTAGTGACAAAACTCACACGTCCCCACCGTCCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAAGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTGCACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGAGCTGCGCCGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCGTGAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA
SEQ ID NO：59
DNA序列22：(IL-15Rα-sushi(73)-Fc-Hole，编码蛋白序列34前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGCGACCCTGCCCTGGTTCACCAACGC
Figure PCTCN2014094947-appb-000031
Figure PCTCN2014094947-appb-000032
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGACCAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：60
DNA序列23：(IL-15Rα-sushi(65)-Fc-Hole，编码蛋白序列35前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGC
Figure PCTCN2014094947-appb-000033
Figure PCTCN2014094947-appb-000034
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAG AGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGACCAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG
SEQ ID NO：61
DNA序列24：(IL-15Rα-sushi(86)-Fc-Hole，编码蛋白序列36前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCAGCGCCACCATCCACAGTAACCACTGCAGGCGTGACC
Figure PCTCN2014094947-appb-000035
Figure PCTCN2014094947-appb-000036
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGACCAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：62
DNA序列25：(IL-15Rα-sushi(102)-Fc-Hole，编码蛋白序列37前体的核苷酸序列):
ATGGACATGCGGGTGCCAGCCCAGCTGCTGGGCCTGTTGCTGCTGTGGTTCC CCGGCTCTCGGTGCATCACCTGCCCTCCACCTATGTCCGTGGAACACGCAGACATCTGGGTCAAGAGCTACAGCTTGTACTCCCGCGAGCGCTACATTTGTAACTCTGGTTTCAAGCGTAAAGCCGGCACCTCCAGCCTGACCGAGTGCGTGTTGAACAAGGCCACCAATGTCGCCCACTGGACAACCCCAAGTCTCAAATGCATTCGCGACCCTGCCCTGGTTCACCAACGCCCAGCGCCACCATCCACAGTAACCACTGCAGGCGTGACCCCACAGCCAGAGAGCCTCTCCCCTTCTGGCAAAGAGC CAGCAGCTTCA
Figure PCTCN2014094947-appb-000037
GAACCTAAGTCCTCTGATAAGACCCACACATGTCCCCCCTGCCCAGCTCCTGAGCTCTTGGGCGGACCTTCCGTGTTTCTGTTCCCCCCAAAGCCCAAGGATACCCTTATGATCAGCAGAACACCCGAAGTTACTTGCGTGGTCGTGGACGTTTCTCACGAAGATCCTGAAGTGAAATTCAACTGGTACGTGGATGGCGTGGAGGTGCACAATGCTAAGACTAAGCCCCGTGAAGAGCAGTACAACTCTACCTACCGGGTCGTTTCAGTGCTGACTGTTCTCCATCAGGACTGGCTCAACGGGAAGGAGTATAAGTGCAAGGTGTCTAACAAGGCACTGCCCGCACCCATCGAGAAGACCATTTCTAAGGCCAAGGGTCAACCACGGGAGCCACAGGTTTACACATTGCCTCCCAGTCGGGAGGAGATGACAAAGAATCAAGTGTCACTTACATGTCTTGTGAAGGGCTTCTACCCCTCAGACATCGCCGTGGAGTGGGAGAGCAACGGACAACCAGAAAACAACTACAAGACCACACCTCCTGTGCTCGATTCAGATGGTTCCTTTTTCTTGACCAGCAAACTCACCGTTGACAAGAGTCGGTGGCAGCAAGGAAATGTGTTCAGCTGTTCTGTGATGCACGAGGCCCTGCACAACCATTATACCCAAAAATCTCTCAGCCTTTCTCCCGGCAAG SEQ ID NO：63
实施例3、蛋白的表达
使用FreeStyle 293细胞(GIBCO,Cat#R79007)瞬时转染表达IL-15蛋白。FreeStyle 293细胞悬浮培养在Freestyle 293expression medium(GIBCO,Cat#12338018),添加终浓度为1％的Ultra Low IgG Fetal Bovine Serum(超低免疫球蛋白胎牛血清，GIBCO,Cat#16250078)。准备实施例1中相关的表达质粒和转染试剂PEI(Polysciences,Cat#239662)，质粒量为100ug/100ml细胞，质粒和PEI的质量比为1:2。转染当天细胞密度为1×106/ml。1L的FreeStyle 293待转染细胞，取50ml Opti-MEM(GIBCO,Cat#11058021)培养基与质粒混匀，静置5min，过滤；另取50ml Opti-MEM培养基与PEI混匀，静置5min，过滤。将质粒和PEI进行混匀，静置15min。将质粒和PEI混合物缓慢加入细胞中，置入37℃，8％CO2，130rpm摇床培养箱中培养。5天后离心收集上清进行蛋白纯化。
实施例4、蛋白的纯化
(1)IL-15异源二聚体蛋白亲和层析法：
细胞培养液经过高速离心后取上清，利用GE的Protein A层析柱进行亲和层析。层析使用平衡缓冲液为1×PBS(pH7.4)，细胞上清上样结合后利用PBS洗涤至紫外线回到基线，然后利用洗脱缓冲液0.1M甘氨酸(pH3.0)洗脱目的蛋白，利用Tris调节pH至中性保存；
(2)IL-15异源二聚体蛋白离子交换层析：
将亲和层析所得产物调节pH至低于或者高于pI 1-2个pH单位，适当稀释以控制样本电导在5ms/cm以下。利用合适的对应pH缓冲液如磷酸缓冲液、醋酸缓冲液等条件，利用本领域内常规的离子交换层析方法如阴离子交换或者阳离子交 换进行对应pH条件下NaCl梯度洗脱，根据SDS-PAGE选择目的蛋白所在的收集管合并保存。
(3)IL-15异源二聚体蛋白体积排阻层析：
将离子交换所得产物超滤浓缩后进行体积排阻层析，如利用GE的Superdex200凝胶进行分离，以去除可能的聚体及其它成分，获得高纯度的目的产物。所得蛋白纯度分析可以通过SDS-PAGE及SEC-HPLC检测进行分析。蛋白浓度通过紫外分光光度法测定。
所得的蛋白序列如实施例1中所述，具体各异源二聚体蛋白由1个或2个选自上述序列的蛋白序列组成，优选经Knob/Hole形式进行配对形成的异源二聚体，经细胞共表达后，分子纯化而得；也可以由未经突变的Fc链区组成双价蛋白。优选实施例如分子3通过融合蛋白IL-15-Fc-Knob和Fc-Hole配对形成(共表达后分子纯化所得)，实施例中的分子7通过融合蛋白IL-15-Fc-Knob和IL-15RαECD-Fc-Hole配对形成(共表达后分子纯化所得)。本发明的非限制性二聚体蛋白的实施如下表1：
表1.各二聚体蛋白设计
编号 蛋白(I) 蛋白(Π)
1 IL-15-Fc(SEQ ID No.8) IL-15-Fc(SEQ ID No.8)
2 Fc-IL-15(SEQ ID No.9) Fc-IL-15(SEQ ID No.9)
3 IL-15-Fc-Knob(SEQ ID No.14) Fc-Hole(SEQ ID No.27)
4 IL-15-Fc-Hole(SEQ ID No.15) Fc-Knob(SEQ ID No.26)
5 Fc-Knob-IL-15(SEQ ID No.16) Fc-Hole(SEQ ID No.27)
6 Fc-Hole-IL-15(SEQ ID No.17) Fc-Knob(SEQ ID No.26)
7 IL-15-Fc–Knob(SEQ ID No.14) IL-15RαECD-Fc-Hole(SEQ ID No.19)
8 IL-15-Fc-Hole(SEQ ID No.15) IL-15RαECD-Fc-Knob(SEQ ID No.18)
9 Fc-Knob-IL-15(SEQ ID No.16) Fc-Hole-IL-15RαECD(SEQ ID No.21)
10 Fc-Hole-IL-15(SEQ ID No.17) Fc-Knob-IL-15RαECD(SEQ ID No.20)
11 IL-15-Fc–Knob(SEQ ID No.14) IL-15Rα-sushi(77)-Fc-Hole(SEQ ID No.23)
12 Fc-Knob(M)-IL-15(SEQ ID No.30) Fc-Hole(M)-IL-15Rα-sushi(65)(SEQ ID No.32)
13 IL-15-Fc-Knob(M)(SEQ ID No.31) IL-15Rα-sushi(65)-Fc-Hole(M)(SEQ ID No.33)
14 IL-15-Fc–Knob(SEQ ID No.14) IL-15Rα-sushi(73)-Fc-Hole(SEQ ID No.34)
15 IL-15-Fc–Knob(SEQ ID No.14) IL-15Rα-sushi(65)-Fc-Hole(SEQ ID No.35)
16 IL-15-Fc–Knob(SEQ ID No.14) IL-15Rα-sushi(86)-Fc-Hole(SEQ ID No.36)
17 IL-15-Fc–Knob(SEQ ID No.14) IL-15Rα-sushi(102)-Fc-Hole(SEQ ID No.37)
表1显示了本发明中涉及的具体的17种二聚体蛋白，分别为二聚体蛋白1-17，其分别由表中相应的蛋白(I)和蛋白(II)重组而成。
测试例
测试例1.体外增殖实验检测
新鲜人外周血单个核细胞(PBMC)在IL-15、本发明的二聚体蛋白1-17作用下的增殖试验。
新鲜PBMC培养在含有10％FBS的RPMI1640培养基中，实验时离心重悬后调整细胞的密度为5×105个/ml，96孔板中每孔加入90ul，样本用PBS按一定倍 数稀释成不同浓度梯度，96孔板中每孔加入10μl，37℃,5％CO2培养箱中培养48小时，取50μl用
Figure PCTCN2014094947-appb-000038
Luminescent Cell Viability Assay试剂盒检测细胞增殖。结果见表2：
表2.体外增殖实验检测结果
样本 PBMC相对活性
IL-15(对照) 100
1 79
2 46
3 367
5 138
7 1100
8 183
9 26
10 11
11 1392
12 210
13 206
14 1243
15 1338
16 1024
17 2677
实施例实验结果：人PBMC增殖实验，结果显示本发明的异源二聚体蛋白蛋白药物3、7、11及14-17增殖效果要明显优于IL-15对照，其增殖效果也明显优于同源二聚体1和2。并且Fc变体在融合蛋白C端的效果好于Fc变体在融合蛋白N端的效果。另外形式的变体分子异源二聚体蛋白12和13增殖效果也优于IL-15对照。
测试例2.IL-15异源二聚体体内PK检测
一、实验目的
评价IL-15异源二聚体蛋白3和7在小鼠体内的药代动力学变化。
二、材料与实验方法
1、受试化合物即加样样本
IL-15，IL-15的异源二聚体蛋白3和7。
2、受试动物
C57BL/6小鼠，SPF，15-16g，♂，购自上海西普尔·必凯实验动物有限责任公司。
3、动物试验方法
27只C57BL/6小鼠，分为3组，每组9只，每笼3只，每个时间点采3只，循环采血。腹腔注射2μg IL-15、10μg二聚体蛋白3和20μg二聚体蛋白7(IL-15、二聚体蛋白3和二聚体蛋白7等摩尔，均为0.15nmol)。给药后30min、1、2、4、8、24、48、72、120h取血，每个点眼眶取血50-100μl。取血清进行人IL-15ELISA检测。
三、实验结果与讨论
IL-15、二聚体蛋白3和二聚体蛋白7等摩尔给药后，IL-15在30分钟内达到最大值，然后随时间被快速代谢，到24小时被全部代谢。二聚体蛋白3在给药后2小时达到最大值，随后随着时间被缓慢代谢，直到120小时被全部代谢。二聚体蛋白7也是在给药后2小时达到最大值，随后随时间被缓慢代谢，到120小时仍能检测到较大浓度的蛋白。
PK实验(图1)显示，二聚体蛋白3和二聚体蛋白7给药后血清中最大蛋白摩尔浓度小于IL-15，但在血清中的保留时间超过IL-15，具有明显的长效作用。
IL-15体内药效检测
本实施例在肺转移模型、裸鼠荷瘤模型和重度联合免疫缺陷NOD-SCID鼠三种模型中，检测IL-15二聚体蛋白3和二聚体蛋白7的药效结果。结果见以下测试例。
测试例3.肺转移模型
一、实验目的
利用B16F10细胞建立小鼠肺转移模型，评价IL-15药物给药后对肿瘤转移和生长的影响。
二、材料与实验方法
1、受试蛋白
IL-15二聚体蛋白3和二聚体蛋白7。
2、受试动物
C57BL/6小鼠，SPF，10-16g，♂，购自上海西普尔·必凯实验动物有限责任公司。
3、动物试验方法
给药方案：32只C57BL/6小鼠，分为4组，每组8只。通过尾静脉注射1.5×105个B16F10细胞，在第1、2、10天腹腔注射PBS、2μg IL-15、11μg二聚体 蛋白3和14μg二聚体蛋白7(IL-15、二聚体蛋白3和二聚体蛋白7等摩尔，均为0.16nmol)。
在第21天处死小鼠，取出肺部，称重，观察肺部黑色肿瘤块，拍照，固定在中性甲醛中，计数黑色肿瘤块数量。
三、实验结果
给药方案中，PBS组小鼠肺部有大量转移生长的黑色素瘤(175±23)；IL-15组肺部有大量的黑色素瘤块(157±20)，约为PBS组的90％；二聚体蛋白3给药组肺部可见少量转移的黑色素瘤块(26±6)，约为PBS组的15％；二聚体蛋白7给药组肺部可见较多转移的黑色素瘤块(83±28)，约为PBS组的49％，PBS组肺部肿瘤块数量明显高于二聚体蛋白3和二聚体蛋白7组，与IL-15组相比没有显著差别。IL-15组肺部肿瘤块数量明显高于二聚体蛋白3组。二聚体蛋白7组的肺部肿瘤块数量明显高于二聚体蛋白3组，如图2所示。PBS组肺部相对重量明显高于二聚体蛋白3和二聚体蛋白7给药组，但与IL-15组相比没有显著差别。IL-15组肺部相对重量明显高于二聚体蛋白3和二聚体蛋白7组，如图3所示。
综上所述，在B16F10小鼠模型中，3种蛋白的药效为二聚体蛋白3＞二聚体蛋白7＞IL-15。
测试例4.裸鼠荷瘤模型
一、实验目的
利用HCT-116(人结肠癌)细胞建立裸鼠荷瘤模型，评价IL-15药物给药后对肿瘤生长的影响。
二、材料与实验方法
1、受试蛋白
IL-15二聚体蛋白3和二聚体蛋白7。
2、受试动物
BALB/cA-nude裸小鼠，SPF，16-20g，♀，购自上海西普尔·必凯实验动物有限责任公司。
3、动物试验方法
(1)裸小鼠实验室环境适应10天。
(2)肿瘤细胞移植
裸小鼠右肋部皮下接种HCT-116细胞(5×106/只)，肿瘤生长20天，长至100±15mm3后将动物随机分组(d0)，每组6只。
(3)给药剂量及方法
腹腔注射给药，每2天给药一次，每周给药3次，IL-15为2μg/只，二聚体蛋白3为10μg/只，二聚体蛋白7为20μg/只(IL-15、二聚体蛋白3和二聚体蛋白7为等摩尔，均为0.15nmol)。
(4)移植瘤体积及裸小鼠体重测定
每周测2-3次瘤体积(图4)，称体重，记录数据。第27天处死，取瘤。
(5)数据统计
使用Excel统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
相对体积(RTV)＝VT/V0
抑瘤率(％)＝(CRTV-TRTV)/CRTV(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。CRTV、TRTV分别为实验结束时的空白对照组(Blank)及实验组的相对肿瘤体积。
三、实验结果
IL-15蛋白对HCT-116肿瘤的生长抑制作用如表3和图4所示，IL-15、3和7等摩尔连续给药27天，每2天给药一次后，IL-15、二聚体蛋白3、二聚体蛋白7组均能抑制HCT-16细胞移植瘤生长，抑制率分别为32％、45％、20％，但由于个体差异较大，与空白对照组相比没有显著性。给药过程中没有出现小鼠死亡，各组小鼠给药期间体重没有明显下降，提示目前给药剂量没有明显的毒副作用。
给药第27天剥离各组小鼠肿瘤称取重量，如图5所示，图中*表示：与空白结照Blank相比p<0.05，即二聚体蛋白3组与Blank组相比肿瘤重量显著降低，IL-15和二聚体蛋白7组肿瘤重量与Blank组相当。
综上所述，在HCT-116裸鼠模型中，3种蛋白的抑瘤效果为二聚体蛋白3＞IL-15＞二聚体蛋白7。
表3.给药蛋白对HCT-116裸小鼠移植瘤的疗效
Figure PCTCN2014094947-appb-000039
测试例5.重度联合免疫缺陷NOD-SCID鼠
一、实验目的
采用HCT-116细胞和人外周血单核细胞(PBMC)在体外混合，接种重度联合免疫缺陷NOD-SCID鼠，评价IL-15给药后对肿瘤生长的影响。Nod-SCID小鼠理论上自身缺乏T细胞和NK细胞，所以用IL-15给药后可以激活人的PBMC细 胞(包括T和NK细胞)杀死肿瘤细胞，抑制肿瘤细胞生长。Nod-SCID小鼠模型更接近人的免疫系统杀死肿瘤细胞。
二、材料与实验方法
1、受试蛋白
IL-15购自近岸蛋白质科技有限公司；
IL-15二聚体蛋白3和7。
2、受试动物
NOD SCID雌性小鼠购自北京维通利华(批号：11400700006527)，6-8周龄，体重20g左右，每个药物组5只。
3、动物试验方法
(1)SCID鼠实验室环境适应10天。
(2)PBMC细胞分离
无菌转移6ml淋巴细胞分离液(Lymphocyte Separation Medium，LSM)到15ml离心管中(之前轻轻颠倒瓶子使LSM充分混合)；
将用肝素抗凝处理的静脉血4ml与同体积生理盐水混合，仔细将稀释血液8ml加入离心管中的LSM(室温，缓慢加入，在血液和LSM中形成一个明显的分层，不要将稀释血液混合入LSM中)；
室温下400g离心15-30分钟，离心可以沉淀红细胞和多核白细胞同时可以在LSM上形成一层单核淋巴细胞；
吸出淋巴细胞上方4-6cm的血浆；
吸取淋巴细胞层以及它下面一半的LSM转移到另外的一个离心管。加入等体积的平衡盐缓冲液PBS，室温1000rpm离心10分钟；
用PBS缓冲液或RPMI-1640培养基清洗细胞，用RPMI-1640培养基重悬细胞；
37℃培养3-4小时，收集悬浮细胞，计数。
(3)肿瘤细胞移植
将HCT-116细胞和PBMC细胞按照4:1比例混合均匀，SCID鼠右肋部皮下接种混合细胞细胞(HCT-116细胞：5×106/只，PBMC：1.25×106/只)，肿瘤生长28天。
(4)给药剂量及方法
接种HCT-116+PBMC细胞后第二天开始腹腔注射给药，每2天给药一次，连续给药10次，IL-15为2μg/只，3为10μg/只，7为20μg/只(IL-15、3和7为等摩尔，均为0.15nmol)。
(5)移植瘤体积及裸小鼠体重测定
从第12天开始每2-3天瘤体积，称体重，记录数据。第28天处死，取瘤。
(6)数据统计
使用Excel统计软件：平均值以avg计算；SD值以STDEV计算；SEM值以 STDEV/SQRT计算；组间差异P值以TTEST计算。
肿瘤体积(V)计算公式为：V＝1/2×L长×L短 2
抑瘤率(％)＝(VT-V0)/VT(％)
其中V0、VT分别为实验开始时及实验结束时的肿瘤体积。
三、实验结果
接种细胞后第二天开始IL-15、二聚体蛋白3和二聚体蛋白7等摩尔连续给药20天，每2天给药1次。IL-15蛋白对HCT-116+PBMC肿瘤生长的抑制作用如表4和图6所示，第1周各组均没有发现肿瘤。从第12天(D12)开始，各组均出现肿瘤，且IL-15、二聚体蛋白3、二聚体蛋白7组肿瘤体积小于PBMC组。到第28天(D28)，IL-15、二聚体蛋白3组能明显抑制HCT-16细胞移植瘤生长，抑制率分别为42％、41％；二聚体蛋白7组抑制率为27％，但与PBMC组相比没有显著性。给药过程中没有出现小鼠死亡，各组小鼠给药期间体重没有明显下降，提示目前给药剂量没有明显的毒副作用。
给药第28天剥离各组小鼠肿瘤称取重量，如图7所示，二聚体蛋白3组与PBMC组相比肿瘤重量显著降低；IL-15组肿瘤重量比PBMC组降低，但没有显著性；二聚体蛋白7组肿瘤重量与PBMC组相当。
综上所述，在HCT-116+PBMC SCID鼠模型中，3种蛋白的抑瘤效果为3≥IL-15＞7。
表4.给药蛋白对HCT-116+PBMC SCID鼠移植瘤的疗效
Figure PCTCN2014094947-appb-000040
表中*表示：p<0.05,vs PBMC。
测试例6体外Mo7e细胞增殖实验
1.主要实验材料
Mo7e(人巨核细胞白血病细胞株)购自于协和医学院；
IL-15购自Novoprotein，货号C016，IL-15类似物(二聚体蛋白11-17)来自内部制备；
Cell Counting Kit-8(CCK-8)，购自WST，货号EX660；
GM-CSF购自NOVOProtein，货号CC79。
2.实验方法
1)Mo7e用改良型RPMI-1640培养基(含2.05mM L-谷氨酰胺，10％FBS和15ng/ml GM-CSF)在37℃(5％CO2)细胞培养箱中培养；
2)将培养状态良好的Mo7e细胞150×g，室温离心5min，弃上清；
3)细胞沉淀用无GM-CSF的培养基洗涤两遍后计数；
4)调整细胞浓度铺96孔板，细胞数量2×104/孔，体积90μl(无GM-CSF)，置于细胞培养箱中静置培养；
5)IL-15及其类似物(二聚体蛋白11-17)分别用PBS作四倍倍比稀释，在96孔板细胞培养2小时后加入细胞培养体系中，10μl/孔，每个受试物浓度做三重复并设空白对照孔(只加PBS)；
6)细胞培养板在培养箱中继续培养3天；
7)所有检测孔中加入10ul CCK-8，培养箱中孵育3h；
8)检测450nm下的吸光度(OD450)。
样品 EC50(nM)相对活性
IL-15(对照) 100.00
11 951.57
12 186.96
13 400.38
14 2526.19
15 2496.47
16 2988.73
17 2060.19
在Mo7e增殖实验中待测分子二聚体蛋白11-17均明显优于IL15对照分子。
测试例7小鼠肺转移模型
1.动物试验方法
32只C57BL/6小鼠(SPF，上海西普尔·必凯实验动物有限责任公司)，通过尾静脉注射1.5×105个B16F10细胞(中科院上海生命科学研究院细胞资源中心，货号TCM36)，分为4组，每组8只。在第1天腹腔注射PBS、2μg IL-15，4.2μg、12.5μg二聚体蛋白17，每2-3天称重一次，在第14天每组各处死1只，观察肺转移情况。在第16天处死全部小鼠，取出肺部，称重，观察肺部黑色肿瘤斑点，拍照，固定在甲醇中，计数黑色肿瘤斑点数量。
2.实验结果
PBS组小鼠肺部有大量转移生长的黑色素瘤(73±43)。IL-15给药组肺部可见大量转移的黑色素瘤斑点(65±29)，为PBS组的90％。二聚体蛋白17-4.2μg给药组肺部可见部分转移的黑色素瘤斑点(32±24)，为PBS组的44％；二聚体蛋白17-12.5μg给药组肺部可见少数转移的黑色素瘤斑点(14±14)，为PBS组的19％。
在本次的B16F10小鼠模型中，二聚体蛋白17的药效明显优于IL-15，如图8表示各组试验小鼠的肺部转移肿瘤斑点数量，与PBS组相比，*表示p<0.05，**表示p<0.01。
PBS组肺部相对重量高于二聚体蛋白17给药组，如图9所示。
二聚体蛋白17-12.5ug剂量组小鼠在给药后第5天体重略有降低，然后逐渐恢复，如图10所示。
综上，二聚体蛋白17能够抑制B16F10细胞在小鼠体内的肺部转移，并且具有剂量效应。

Claims (22)

  1. 一种IL-15异源二聚体蛋白，其包括：
    蛋白(I)，和蛋白(Π)；
    其中
    蛋白(I)由IL-15或其变体和第一Fc变体重组而成；
    蛋白(Π)是第二Fc变体，或者蛋白(Π)由IL-15Rα或其变体和第二Fc变体重组而成；
    蛋白(I)和蛋白(Π)通过第一Fc变体和第二Fc变体之间的相互作用形成稳定的异源二聚体蛋白。
  2. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的IL-15序列为SEQ ID NO：1。
  3. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的蛋白(Π)是第二Fc变体。
  4. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的蛋白(Π)由IL-15Rα或其变体和第二Fc变体重组而成。
  5. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的IL-15Rα变体为IL-15Rα胞外域部分或其功能性片段，所述的功能性片段优选为IL-15Rα胞外域部分65-120个氨基酸的缩短形式，更优选为65-102个氨基酸的缩短形式。
  6. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的IL-15Rα变体的序列选自SEQ ID NO：2-7，更优选选自SEQ ID NO：3-7。
  7. 如权利要求1所述的IL-15异源二聚体蛋白，其中：
    所述的第一Fc变体选自Knob修饰的Fc和Hole修饰的Fc；或所述的第二Fc变体选自Hole修饰的Fc和Knob修饰的Fc；
    当第一Fc变体是Knob修饰的Fc时，第二Fc变体是Hole修饰的Fc；或者
    当第二Fc变体是Knob修饰的Fc时，第一Fc变体是Hole修饰的Fc；
    优选的，所述的第一Fc变体和第二Fc变体连接在蛋白IL-15和IL-15Rα的C端。
  8. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述的第一Fc变体的序列选自SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29； 和其中所述的第二Fc变体的序列选自SEQ ID NO：26、SEQ ID NO：27、SEQ ID NO：28和SEQ ID NO：29。
  9. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述蛋白(I)的序列选自SEQ ID NO：14-17，优选为SEQ ID NO：14；所述蛋白(Π)的序列选自SEQ ID NO：18-25、34-37，优选选自SEQ ID NO：23、34-37，更优选选自SEQ ID NO：34-37。
  10. 如权利要求1所述的IL-15异源二聚体蛋白，其中所述蛋白(I)的序列选自SEQ ID NO：30-31；所述蛋白(Π)的序列选自SEQ ID NO：32-33。
  11. 如权利要求1所述的IL-15异源二聚体蛋白，其选自如下的二聚体蛋白3-17，其中所述的二聚体蛋白3-17分别由相对应的蛋白(I)和蛋白(Π)重组而成：
    Figure PCTCN2014094947-appb-100001
  12. 一种核酸，其编码如权利要求1-11所述的IL-15异源二聚体蛋白。
  13. 一种DNA载体，其包括权利要求12所述的核酸。
  14. 一种宿主细胞，其转染有权利要求13所述的DNA载体。
  15. 一种制备权利要求1-11任一项所述的IL-15异源二聚体蛋白的方法，所述方法包括：
    在足以表达权利要求1-11所述的IL-15异源二聚体蛋白的条件下，培养权利要求14所述的宿主细胞；表达并纯化所述的IL-15异源二聚体蛋白。
  16. 一种药物组合物，其含有：如权利要求1至11任一项所述的IL-15异源二聚体蛋白、和可药用的赋形剂、稀释剂或载体。
  17. 一种靶向性蛋白分子，其含有权利要求1至11任一项所述的IL-15异源二聚体蛋白结构。
  18. 一种用于刺激或抑制哺乳动物免疫应答的方法，所述方法包括：
    向所述哺乳动物给予有效量的权利要求1-11任一项所述的IL-15异源二聚体蛋白，或权利要求16所述的药物组合物，或权利要求17所述的靶向性蛋白分子。
  19. 如权利要求1至11任一项所述的IL-15异源二聚体蛋白、如权利要求16所述的药物组合物，或如权利要求17所述的靶向性蛋白分子，在制备用于治疗IL-15介导的疾病或病症的药物中的用途。
  20. 如权利要求19所述的用途，其中：
    所述的疾病或病症选自传染病、癌症、血液病、炎性疾病和自身免疫性疾病；
    所述的癌症选自黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌；
    所述的传染病选自天花病毒感染、HIV感染、细菌感染、真菌感染、HBV感染；
    所述的血液病选自贫血、急性髓系白血病、骨髓增生异常综合征、T-细胞大颗粒淋巴细胞性白血病；
    所述的自身免疫性疾病选自多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
  21. 如权利要求19所述的用途，其中所述如权利要求1至11任一项所述的IL-15蛋白复合物、或如权利要求16所述的药物组合物，或如权利要求17所述的靶向性蛋白分子与小分子抑制剂或抗体类药物联合施用；所述的小分子抑制剂选自烷化剂；所述的的抗体类药物选自单克隆抗体药物，更优选抗CD20、PD1、PDL1、Her2抗体。
  22. 如权利要求1至11任一项所述的IL-15蛋白复合物、或如权利要求16所述的药物组合物，或如权利要求17所述的靶向性蛋白分子，在制备用于细胞免疫治疗的药物中的用途，尤其是在制备用于DC、CIK、DC-CIK、ECIK、NK、CAS-T、BiAb-T、TCR-T、CAR-T肿瘤免疫细胞治疗中的药物的用途。
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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106496331A (zh) * 2016-11-08 2017-03-15 北京启康兴业生物医药科技有限公司 一种新型FSH‑Fc融合蛋白及其制备方法和用途
WO2018068257A1 (zh) * 2016-10-13 2018-04-19 北京艺妙神州医疗科技有限公司 通用型car-t细胞及其制备方法和用途
WO2018121605A1 (zh) * 2016-12-29 2018-07-05 天津天锐生物科技有限公司 一种多功能蛋白质
WO2019057180A1 (en) * 2017-09-25 2019-03-28 Dingfu Biotarget Co., Ltd. PROTEIN HETÉRODIMÈRE AND USE THEREOF
WO2019057181A1 (en) * 2017-09-25 2019-03-28 Dingfu Biotarget Co., Ltd. METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER
WO2019143669A1 (en) * 2018-01-18 2019-07-25 Nant Holdings Ip, Llc Fusion protein extensions
JP2019533444A (ja) * 2016-10-14 2019-11-21 ゼンコア インコーポレイテッド ＩＬ−１５／ＩＬ−１５ＲアルファＦｃ融合タンパク質およびＰＤ−１抗体の断片を含む二重特異性ヘテロ二量体融合タンパク質
WO2019229658A1 (en) 2018-05-30 2019-12-05 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
EP3430055B1 (en) * 2016-03-17 2020-10-28 Oslo Universitetssykehus HF Fusion proteins targeting tumour associated macrophages for treating cancer
WO2021053559A1 (en) 2019-09-18 2021-03-25 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
WO2021054867A1 (ru) 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Иммуноцитокин, включающий гетеро димерный белковый комплекс на основе il-15 и il-15rα
WO2021119429A1 (en) * 2019-12-13 2021-06-17 Cugene Inc. Novel interleukin-15 (il-15) fusion proteins and uses thereof
US11084863B2 (en) 2017-06-30 2021-08-10 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
WO2021253360A1 (en) * 2020-06-18 2021-12-23 Proviva Therapeutics (Hong Kong) Limited Activatable procytokines
WO2022000136A1 (en) * 2020-06-28 2022-01-06 Dingfu Biotarget Co., Ltd. Methods and compositions for cancer treatment
US11377477B2 (en) 2018-10-12 2022-07-05 Xencor, Inc. PD-1 targeted IL-15/IL-15RALPHA fc fusion proteins and uses in combination therapies thereof
US11505595B2 (en) 2018-04-18 2022-11-22 Xencor, Inc. TIM-3 targeted heterodimeric fusion proteins containing IL-15/IL-15RA Fc-fusion proteins and TIM-3 antigen binding domains
US11524991B2 (en) 2018-04-18 2022-12-13 Xencor, Inc. PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
WO2023017191A1 (en) 2021-08-13 2023-02-16 Cytune Pharma Il-2/il-15rbetagamma agonist combination with antibody-drug conjugates for treating cancer
US11618776B2 (en) 2018-12-20 2023-04-04 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15/IL-15RA and NKG2D antigen binding domains
CN116574192A (zh) * 2023-03-30 2023-08-11 上海妙聚生物科技有限公司 一种可条件性释放并激活的细胞因子融合蛋白及其制备和用途
WO2024037322A1 (zh) * 2022-08-15 2024-02-22 海徕科(北京)生物技术有限公司 一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途
US11932675B2 (en) 2019-10-11 2024-03-19 Genentech, Inc. PD-1 targeted IL-15/IL-15Rα Fc fusion proteins with improved properties
US12139525B2 (en) 2021-07-02 2024-11-12 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains

Families Citing this family (61)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10851178B2 (en) 2011-10-10 2020-12-01 Xencor, Inc. Heterodimeric human IgG1 polypeptides with isoelectric point modifications
JP6618362B2 (ja) 2013-01-14 2019-12-11 ゼンコア インコーポレイテッド 新規異種二量体タンパク質
US11053316B2 (en) 2013-01-14 2021-07-06 Xencor, Inc. Optimized antibody variable regions
US10968276B2 (en) 2013-03-12 2021-04-06 Xencor, Inc. Optimized anti-CD3 variable regions
US10487155B2 (en) 2013-01-14 2019-11-26 Xencor, Inc. Heterodimeric proteins
US9605084B2 (en) 2013-03-15 2017-03-28 Xencor, Inc. Heterodimeric proteins
US9701759B2 (en) 2013-01-14 2017-07-11 Xencor, Inc. Heterodimeric proteins
US10106624B2 (en) 2013-03-15 2018-10-23 Xencor, Inc. Heterodimeric proteins
US10519242B2 (en) 2013-03-15 2019-12-31 Xencor, Inc. Targeting regulatory T cells with heterodimeric proteins
DK2970486T3 (en) 2013-03-15 2018-08-06 Xencor Inc MODULATION OF T-CELLS WITH BISPECIFIC ANTIBODIES AND FC-FUSIONS
US10858417B2 (en) 2013-03-15 2020-12-08 Xencor, Inc. Heterodimeric proteins
PE20161431A1 (es) 2014-03-28 2017-01-22 Xencor Inc Anticuerpos biespecificos que se unen a cd38 y cd3
TN2017000223A1 (en) 2014-11-26 2018-10-19 Xencor Inc Heterodimeric antibodies that bind cd3 and tumor antigens
US10259887B2 (en) 2014-11-26 2019-04-16 Xencor, Inc. Heterodimeric antibodies that bind CD3 and tumor antigens
US10526417B2 (en) 2014-11-26 2020-01-07 Xencor, Inc. Heterodimeric antibodies that bind CD3 and CD38
US10905743B2 (en) * 2014-12-19 2021-02-02 Jiangsu Hengrui Medicine Co., Ltd. Interleukin 15 protein complex
WO2016105450A2 (en) 2014-12-22 2016-06-30 Xencor, Inc. Trispecific antibodies
US10227411B2 (en) 2015-03-05 2019-03-12 Xencor, Inc. Modulation of T cells with bispecific antibodies and FC fusions
WO2017100372A1 (en) 2015-12-07 2017-06-15 Xencor, Inc. Heterodimeric antibodies that bind cd3 and psma
AU2017268399B2 (en) 2016-05-18 2023-01-12 Modernatx, Inc. mRNA combination therapy for the treatment of cancer
CA3029813A1 (en) 2016-06-13 2017-12-21 Torque Therapeutics, Inc. Methods and compositions for promoting immune cell function
US10787518B2 (en) 2016-06-14 2020-09-29 Xencor, Inc. Bispecific checkpoint inhibitor antibodies
KR20190020341A (ko) 2016-06-28 2019-02-28 젠코어 인코포레이티드 소마토스타틴 수용체 2에 결합하는 이종이량체 항체
US10793632B2 (en) 2016-08-30 2020-10-06 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
CN108250301A (zh) * 2016-12-29 2018-07-06 天津天锐生物科技有限公司 一种多靶点嵌合抗原受体
US11629340B2 (en) 2017-03-03 2023-04-18 Obsidian Therapeutics, Inc. DHFR tunable protein regulation
CN109963581B (zh) * 2017-07-25 2023-09-19 江苏恒瑞医药股份有限公司 一种il-15蛋白复合物药物组合物及其用途
US11524033B2 (en) 2017-09-05 2022-12-13 Torque Therapeutics, Inc. Therapeutic protein compositions and methods of making and using the same
JP2021502100A (ja) 2017-11-08 2021-01-28 ゼンコア インコーポレイテッド 新規抗ｐｄ−１配列を用いた二重特異性および単一特異性抗体
US10981992B2 (en) 2017-11-08 2021-04-20 Xencor, Inc. Bispecific immunomodulatory antibodies that bind costimulatory and checkpoint receptors
EP3717008A1 (en) 2017-12-01 2020-10-07 Merus N.V. Use of bispecific antibody and il-15 for combination therapy
JP2021506291A (ja) 2017-12-19 2021-02-22 ゼンコア インコーポレイテッド 改変されたｉｌ−２ ｆｃ融合タンパク質
CN113166220A (zh) * 2018-03-09 2021-07-23 奥美药业有限公司 创新细胞因子前药
US11267866B2 (en) 2018-04-04 2022-03-08 Nant Holdings Ip, Llc Immunoglobulin complex comprising interleukin-15
WO2019195623A2 (en) 2018-04-04 2019-10-10 Xencor, Inc. Heterodimeric antibodies that bind fibroblast activation protein
KR20210003813A (ko) * 2018-04-18 2021-01-12 젠코어 인코포레이티드 IL-15/IL-15Rα Fc-융합 단백질 및 LAG-3 항원 결합 도메인을 함유하는 LAG-3 표적화 이종이량체 융합 단백질
AU2019255744A1 (en) * 2018-04-18 2020-11-26 Xencor, Inc. IL-15/IL-15Ra heterodimeric Fc fusion proteins and uses thereof
AU2019288471A1 (en) * 2018-06-22 2021-01-14 Cugene Inc. Novel interleukin-15 (1L-15) fusion proteins and uses thereof
WO2019246392A1 (en) 2018-06-22 2019-12-26 Cugene Inc. Cytokine-based bioactivatable drugs and methods of uses thereof
CN113286812A (zh) * 2018-09-27 2021-08-20 西里欧发展公司 掩蔽型细胞因子多肽
WO2020072821A2 (en) 2018-10-03 2020-04-09 Xencor, Inc. Il-12 heterodimeric fc-fusion proteins
MX2021010390A (es) 2019-03-01 2021-11-17 Xencor Inc Anticuerpos heterodimericos que se unen a enpp3 y cd3.
WO2020192648A1 (en) * 2019-03-25 2020-10-01 Dingfu Biotarget Co., Ltd. Proteinaceous heterodimer and use thereof
US20220241375A1 (en) 2019-05-20 2022-08-04 Cytune Pharma Il-2/il-15r beta gamma agonist dosing regimens for treating cancer or infectious diseases
AU2020291012A1 (en) 2019-06-12 2021-12-16 AskGene Pharma, Inc. Novel IL-15 prodrugs and methods of use thereof
CN112679615B (zh) * 2019-10-17 2023-09-22 瑞阳(苏州)生物科技有限公司 一种融合蛋白
AU2020372413A1 (en) 2019-10-23 2022-06-09 Cue Biopharma, Inc. TGF-beta polypeptides
CN113135996A (zh) * 2019-12-09 2021-07-20 启愈生物技术(上海)有限公司 一种双特异抗体及其应用
JP2023520514A (ja) * 2020-04-01 2023-05-17 エクシリオ デベロップメント， インコーポレイテッド マスク型ｉｌ－１５サイトカインおよびその切断産物
US11919956B2 (en) 2020-05-14 2024-03-05 Xencor, Inc. Heterodimeric antibodies that bind prostate specific membrane antigen (PSMA) and CD3
CN111848813A (zh) * 2020-06-04 2020-10-30 泰州市百英生物科技有限公司 一种IL7-Fc-GMCSF融合蛋白及其应用
EP4200332A1 (en) 2020-08-19 2023-06-28 Xencor, Inc. Anti-cd28 and/or anti-b7h3 compositions
MX2023004879A (es) 2020-10-26 2023-05-11 Cytune Pharma AGONISTA DE IL-2/IL-15RßY PARA EL TRATAMIENTO DEL CARCINOMA DE CÉLULAS ESCAMOSAS.
KR20230096047A (ko) 2020-10-26 2023-06-29 싸이튠 파마 비흑색종 피부암 치료용 IL-2/IL-15Rβγ 작용제
KR20230156079A (ko) 2021-03-09 2023-11-13 젠코어 인코포레이티드 Cd3과 cldn6에 결합하는 이종이량체 항체
WO2022192586A1 (en) 2021-03-10 2022-09-15 Xencor, Inc. Heterodimeric antibodies that bind cd3 and gpc3
BR112023021886A8 (pt) * 2021-04-22 2024-01-09 Guangdong Fapon Biopharma Inc Polipeptídeo de fusão biespecífico multifuncional
WO2023046093A1 (zh) * 2021-09-24 2023-03-30 广东菲鹏制药股份有限公司 一种双特异性多肽复合物
WO2023086772A1 (en) 2021-11-12 2023-05-19 Xencor, Inc. Bispecific antibodies that bind to b7h3 and nkg2d
WO2024102636A1 (en) 2022-11-07 2024-05-16 Xencor, Inc. Bispecific antibodies that bind to b7h3 and mica/b
WO2024215989A1 (en) 2023-04-14 2024-10-17 Sotio Biotech Inc. ENGINEERED IMMUNE CELLS FOR TREATING CANCER IN COMBINATION WITH IL-2/IL-15 RECEPTOR βγ AGONISTS

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
CN1760209A (zh) * 2004-10-15 2006-04-19 上海海欣生物技术有限公司 人白细胞介素15与Fc融合蛋白
CN1942481A (zh) 2004-04-14 2007-04-04 豪夫迈－罗氏公司 用于制备IL－15/Fc融合蛋白的表达系统及其用途
US20100286374A1 (en) 2008-01-07 2010-11-11 Gunasekaran Kannan Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2012040323A2 (en) * 2010-09-21 2012-03-29 Altor Bioscien Corporation Multimeric il-15 soluble fusion molecules and methods of making and using same
CN101360827B (zh) 2005-10-20 2013-03-20 国立医学与健康研究所 IL-15Rα sushi结构域作为通过IL-15Rβ/γ的IL-15作用的选择性和有效的增强剂以及超激动剂(IL15Rα sushi-IL15)融合蛋白

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5731168A (en) 1995-03-01 1998-03-24 Genentech, Inc. Method for making heteromultimeric polypeptides
ATE516305T1 (de) 2004-02-27 2011-07-15 Inst Nat Sante Rech Med Il-15-bindungsstelle für il-15-ralpha und spezifische il-15-mutanten mit wirkung als agonisten/antagonisten
CU23472A1 (es) * 2004-09-17 2009-12-17 Ct Ingenieria Genetica Biotech Péptido antagonista de la interleucina-15
US20060257361A1 (en) * 2005-04-12 2006-11-16 Government Of The Us, As Represented By The Secretary, Department Of Health And Human Services Novel form of interleukin-15, Fc-IL-15, and methods of use
EP2724727A1 (en) 2007-06-27 2014-04-30 The United States of America, as Represented by The Secretary, Department of Health and Human Services Complexes of IL-15 and IL-15R alpha and uses thereof
EA201892619A1 (ru) * 2011-04-29 2019-04-30 Роше Гликарт Аг Иммуноконъюгаты, содержащие мутантные полипептиды интерлейкина-2
CN102558355B (zh) 2011-12-31 2015-02-25 苏州康宁杰瑞生物科技有限公司 基于电荷网络的异二聚体fc改造方法及异二聚体蛋白的制备方法

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4683195A (en) 1986-01-30 1987-07-28 Cetus Corporation Process for amplifying, detecting, and/or-cloning nucleic acid sequences
US4683195B1 (zh) 1986-01-30 1990-11-27 Cetus Corp
CN1942481A (zh) 2004-04-14 2007-04-04 豪夫迈－罗氏公司 用于制备IL－15/Fc融合蛋白的表达系统及其用途
CN1760209A (zh) * 2004-10-15 2006-04-19 上海海欣生物技术有限公司 人白细胞介素15与Fc融合蛋白
CN100334112C (zh) 2004-10-15 2007-08-29 上海海欣生物技术有限公司 人白细胞介素15与Fc融合蛋白
CN101360827B (zh) 2005-10-20 2013-03-20 国立医学与健康研究所 IL-15Rα sushi结构域作为通过IL-15Rβ/γ的IL-15作用的选择性和有效的增强剂以及超激动剂(IL15Rα sushi-IL15)融合蛋白
US20100286374A1 (en) 2008-01-07 2010-11-11 Gunasekaran Kannan Method for making antibody fc-heterodimeric molecules using electrostatic steering effects
WO2012040323A2 (en) * 2010-09-21 2012-03-29 Altor Bioscien Corporation Multimeric il-15 soluble fusion molecules and methods of making and using same

Non-Patent Citations (10)

* Cited by examiner, † Cited by third party
Title
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", GREENE PUBLISHING ASSOCIATES, WILEY INTERSCIENCE
CATER ET AL., PROTEIN ENGINEERING, vol. 9, no. 7, 1996, pages 617 - 621
J. BIOL. CHEM, vol. 243, 1968, pages 3558
JONATHAN H. DAVIS ET AL., PROTEIN ENGINEERING, DESIGN & SELECTION, 2010, pages 1 - 8
MABS 3, vol. 6, November 2011 (2011-11-01), pages 546 - 557
RIDGWAY, J.B. ET AL.: "Knobs-into-Holes' Engineering of Antibody CH 3 Domains for Heavy Chain Heterodimerization", PROTEIN ENGINEERING, vol. 9, no. 7, 31 July 1996 (1996-07-31), pages 617 - 621, XP002084766 *
SAMBROOK ET AL.: "Molecular Cloning, A Laboratory Manual", COLD SPRING HARBOR LABORATORY
SCIENCE, vol. 317, 2007, pages 5844
See also references of EP3093295A4
STOKLASEK, T.A. ET AL.: "Combined IL -15/ IL -15Ralpha Immunotherapy Maximizes IL -15 Activity In Vivo", THE JOURNAL OF IMMUNOLOGY, vol. 177, no. 9, 1 November 2006 (2006-11-01), pages 6072 - 6080, XP002636847 *

Cited By (35)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3430055B1 (en) * 2016-03-17 2020-10-28 Oslo Universitetssykehus HF Fusion proteins targeting tumour associated macrophages for treating cancer
US11186641B2 (en) 2016-03-17 2021-11-30 Oslo Universitetssykehus Hf Fusion proteins targeting tumour associated macrophages for treating cancer
WO2018068257A1 (zh) * 2016-10-13 2018-04-19 北京艺妙神州医疗科技有限公司 通用型car-t细胞及其制备方法和用途
JP7273453B2 (ja) 2016-10-14 2023-05-15 ゼンコア インコーポレイテッド ＩＬ－１５／ＩＬ－１５ＲアルファＦｃ融合タンパク質およびＰＤ－１抗体の断片を含む二重特異性ヘテロ二量体融合タンパク質
US11584794B2 (en) 2016-10-14 2023-02-21 Xencor, Inc. Bispecific heterodimeric fusion proteins containing IL-15-IL-15Ralpha Fc-fusion proteins and immune checkpoint antibody fragments
JP2019533444A (ja) * 2016-10-14 2019-11-21 ゼンコア インコーポレイテッド ＩＬ−１５／ＩＬ−１５ＲアルファＦｃ融合タンパク質およびＰＤ−１抗体の断片を含む二重特異性ヘテロ二量体融合タンパク質
US10501543B2 (en) 2016-10-14 2019-12-10 Xencor, Inc. IL15/IL15Rα heterodimeric Fc-fusion proteins
US10550185B2 (en) 2016-10-14 2020-02-04 Xencor, Inc. Bispecific heterodimeric fusion proteins containing IL-15-IL-15Rα Fc-fusion proteins and PD-1 antibody fragments
CN106496331A (zh) * 2016-11-08 2017-03-15 北京启康兴业生物医药科技有限公司 一种新型FSH‑Fc融合蛋白及其制备方法和用途
CN106496331B (zh) * 2016-11-08 2020-03-13 北京智岭生物医药科技有限公司 一种FSH-Fc融合蛋白及其制备方法和用途
WO2018121605A1 (zh) * 2016-12-29 2018-07-05 天津天锐生物科技有限公司 一种多功能蛋白质
US11084863B2 (en) 2017-06-30 2021-08-10 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
CN111132998A (zh) * 2017-09-25 2020-05-08 苏州丁孚靶点生物技术有限公司 用于癌症治疗的方法和组合物
US11987609B2 (en) 2017-09-25 2024-05-21 Dingfu Biotarget Co., Ltd. Proteinaceous heterodimer and use thereof
CN111132998B (zh) * 2017-09-25 2024-01-30 苏州丁孚靶点生物技术有限公司 用于癌症治疗的方法和组合物
US11883503B2 (en) 2017-09-25 2024-01-30 Dingfu Biotarget Co., Ltd. Methods and compositions for cancer treatment
WO2019057180A1 (en) * 2017-09-25 2019-03-28 Dingfu Biotarget Co., Ltd. PROTEIN HETÉRODIMÈRE AND USE THEREOF
WO2019057181A1 (en) * 2017-09-25 2019-03-28 Dingfu Biotarget Co., Ltd. METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER
WO2019143669A1 (en) * 2018-01-18 2019-07-25 Nant Holdings Ip, Llc Fusion protein extensions
US11505595B2 (en) 2018-04-18 2022-11-22 Xencor, Inc. TIM-3 targeted heterodimeric fusion proteins containing IL-15/IL-15RA Fc-fusion proteins and TIM-3 antigen binding domains
US11524991B2 (en) 2018-04-18 2022-12-13 Xencor, Inc. PD-1 targeted heterodimeric fusion proteins containing IL-15/IL-15Ra Fc-fusion proteins and PD-1 antigen binding domains and uses thereof
WO2019229658A1 (en) 2018-05-30 2019-12-05 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
US11377477B2 (en) 2018-10-12 2022-07-05 Xencor, Inc. PD-1 targeted IL-15/IL-15RALPHA fc fusion proteins and uses in combination therapies thereof
US11618776B2 (en) 2018-12-20 2023-04-04 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15/IL-15RA and NKG2D antigen binding domains
WO2021053559A1 (en) 2019-09-18 2021-03-25 Novartis Ag Entpd2 antibodies, combination therapies, and methods of using the antibodies and combination therapies
RU2753282C2 (ru) * 2019-09-19 2021-08-12 Закрытое Акционерное Общество "Биокад" ИММУНОЦИТОКИН, ВКЛЮЧАЮЩИЙ ГЕТЕРОДИМЕРНЫЙ БЕЛКОВЫЙ КОМПЛЕКС НА ОСНОВЕ IL-15/IL-15Rα, И ЕГО ПРИМЕНЕНИЕ
WO2021054867A1 (ru) 2019-09-19 2021-03-25 Закрытое Акционерное Общество "Биокад" Иммуноцитокин, включающий гетеро димерный белковый комплекс на основе il-15 и il-15rα
US11932675B2 (en) 2019-10-11 2024-03-19 Genentech, Inc. PD-1 targeted IL-15/IL-15Rα Fc fusion proteins with improved properties
WO2021119429A1 (en) * 2019-12-13 2021-06-17 Cugene Inc. Novel interleukin-15 (il-15) fusion proteins and uses thereof
WO2021253360A1 (en) * 2020-06-18 2021-12-23 Proviva Therapeutics (Hong Kong) Limited Activatable procytokines
WO2022000136A1 (en) * 2020-06-28 2022-01-06 Dingfu Biotarget Co., Ltd. Methods and compositions for cancer treatment
US12139525B2 (en) 2021-07-02 2024-11-12 Xencor, Inc. Targeted heterodimeric Fc fusion proteins containing IL-15 IL-15alpha and antigen binding domains
WO2023017191A1 (en) 2021-08-13 2023-02-16 Cytune Pharma Il-2/il-15rbetagamma agonist combination with antibody-drug conjugates for treating cancer
WO2024037322A1 (zh) * 2022-08-15 2024-02-22 海徕科(北京)生物技术有限公司 一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途
CN116574192A (zh) * 2023-03-30 2023-08-11 上海妙聚生物科技有限公司 一种可条件性释放并激活的细胞因子融合蛋白及其制备和用途

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