WO2022247778A1 - 一种新型白介素15突变体多肽的开发及其应用 - Google Patents

一种新型白介素15突变体多肽的开发及其应用 Download PDF

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WO2022247778A1
WO2022247778A1 PCT/CN2022/094443 CN2022094443W WO2022247778A1 WO 2022247778 A1 WO2022247778 A1 WO 2022247778A1 CN 2022094443 W CN2022094443 W CN 2022094443W WO 2022247778 A1 WO2022247778 A1 WO 2022247778A1
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amino acid
novel
mutant polypeptide
seq
acid sequence
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French (fr)
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纪恩
王春雨
吴艳玲
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苏州复融生物技术有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5443—IL-15
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • G01N33/6869—Interleukin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/52—Assays involving cytokines
    • G01N2333/54—Interleukins [IL]
    • G01N2333/5443—IL-15
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention belongs to the field of biotechnology, and specifically relates to a novel interleukin-15 mutant polypeptide, its preparation method, nucleic acid, plasmid, and host cell required for constructing the novel interleukin-15 mutant polypeptide, corresponding pharmaceutical compositions and applications thereof .
  • Interleukin 15 is a soluble cytokine found in the supernatant of the monkey kidney epidermal cell line CV21/EBNA by Grabstein et al. in 1994. It can maintain the proliferation of CTLL-2 cells because it interacts with IL-2 There are many similar biological activities that have attracted much attention. Both IL-2 and IL-15 belong to the IL-2 family, and IL-2 family member receptors all contain ⁇ c chains: including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21 . Among them, IL-2 and IL-15 have common IL-2/IL-15R ⁇ (CD122) and ⁇ c receptors (CD132).
  • the specific receptor of IL-2 is IL-2R ⁇ (CD25), which is mainly expressed on the surface of activated T cells.
  • IL-2 is the most important cytokine that promotes the proliferation of T cells; the specific receptor of IL-15 is IL15R ⁇ ( CD215).
  • IL-15 can bind to the IL-15 ⁇ / ⁇ receptor on the cell surface and activate the downstream STAT5 signaling pathway, thereby promoting the differentiation and expansion of CD8+ T cells.
  • IL-2 mainly exists in a free state, so it can play a role as long as it binds to cell surface receptors.
  • IL-15 and IL-15R ⁇ are mainly expressed on the surface of DC cells and monocytes, and less in the free state. Therefore, the activation of the IL-15 signaling pathway is more likely to activate downstream signaling pathways to play a role after cell-to-cell contact.
  • IL-15 signaling is through a heterotrimeric complex of IL-15R ⁇ , IL-15R ⁇ and ⁇ c.
  • IL-15 The mechanism of action of IL-15 is transfer and presentation.
  • One cell presents its membrane-bound IL-15/IL-15R ⁇ complex to another target cell expressing IL-15R ⁇ and ⁇ c receptors.
  • This method requires that one cell must be in contact with another cell to produce an effect. This is not only the need for the regulation of the body's immune system, but also restricts the use of IL-15/IL-15R ⁇ complexes to a certain extent. Immunotherapy for related diseases.
  • novel IL-15/IL-15R ⁇ complexes using synthetic biology techniques.
  • the inventors innovatively used synthetic biology in the field of long-acting proteins to develop a novel monomeric Fc based on antibody IgG Fc (CN109705211B; 202011161007.3).
  • the novel interleukin 15 mutant polypeptide of the present application introduces this novel monomeric Fc.
  • using the mechanism of IL-15 transmission and presentation we innovatively developed a new type of interleukin-15 mutant polypeptide that can enhance or reduce the binding or biological activity of IL-15R ⁇ and ⁇ c receptors.
  • the main purpose of this application is to provide a novel IL-15 mutant polypeptide and its application.
  • the present invention provides the following technical solutions:
  • a novel IL-15 mutant polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1, wherein:
  • X1 is selected from any amino acid in A and N;
  • X2 is selected from any amino acid in S, D, Y;
  • X3 is selected from any amino acid in D and F;
  • X4 is selected from D, G, any amino acid in N;
  • X5 is selected from any amino acid in E and Y;
  • X6 is selected from any amino acid in I, Q, and K;
  • X7 is selected from any amino acid in N and Y;
  • X8 is selected from any amino acid in Q, S, F, A;
  • X9 is selected from any amino acid in M and H;
  • X10 is selected from any amino acid in I, A, H;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is A, X2 is S, X3 is D, X4 is D, X5 is E, X6 is I, and X7 is N , X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is selected from any amino acid in D and Y, X3 is D, X4 is D, X5 is E, X6 is I, X7 is N, X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is F, X4 is D, X5 is E, and X6 is I , X7 is N, X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, and X4 is selected from any amino acid in G and N , X5 is E, X6 is I, X7 is N, X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is Y, and X6 is I, X7 is N, X8 is Q, X9 is M, and X10 is I.
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is E, X6 is any amino acid selected from Q and K, X7 is N, X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is E, X6 is I, X7 is Y, X8 is Q, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is E, and X6 is I , X7 is Y, X8 is selected from S, F, any amino acid in A, X9 is M, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is E, and X6 is I , X7 is N, X8 is Q, X9 is H, and X10 is I;
  • the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein X1 is N, X2 is S, X3 is D, X4 is D, X5 is E, and X6 is I, X7 is N, X8 is Q, X9 is M, and X10 is selected from any amino acid in A and H.
  • SEQ ID NO: 1 amino acid sequence of IL-15 mutant
  • SEQ ID NO:2 amino acid sequence of mutant B1-A1
  • SEQ ID NO:4 amino acid sequence of mutant B1-H5
  • SEQ ID NO:10 amino acid sequence of mutant B4-B6
  • SEQ ID NO:12 amino acid sequence of mutant R2-F7
  • SEQ ID NO:16 amino acid sequence of mutant R4-E5
  • the second aspect of the present invention provides a nucleic acid molecule encoding the aforementioned novel IL-15 mutant polypeptide.
  • the third aspect of the present invention provides a plasmid, which contains the above-mentioned nucleic acid molecule.
  • the fourth aspect of the present invention provides a host cell containing the above-mentioned plasmid.
  • a fusion body is provided, and the fusion body is connected by a fusion partner to the N-terminal and/or C-terminal of the above-mentioned novel IL-15 mutant polypeptide;
  • the fusion partner is at least one of heterologous proteins, adhesion molecules, nucleic acid molecules, small molecular compounds, toxins, immune cells, and detectable markers;
  • the heterologous protein is an antibody, an antigen, a cytokine, a soluble receptor domain, a ligand, an enzyme, a peptide or a protein domain; the antigen is derived from an adeno-associated virus;
  • the immune cells are chimeric antigen receptor T cells.
  • the sixth aspect of the present invention provides a pharmaceutical composition, which contains an effective preventive or therapeutic dose of the above-mentioned novel IL-15 mutant polypeptide, or the above-mentioned fusion, and a pharmaceutically acceptable carrier .
  • the seventh aspect of the present invention provides the use of the above-mentioned IL-15 mutant polypeptide and the above-mentioned pharmaceutical composition in the preparation of a drug for biological material treatment.
  • the eighth aspect of the present invention provides a method for treating autoimmune disease, inflammatory disease, neurodegenerative disease, cancer or pathogenic infection, comprising administering to the subject an effective therapeutic amount of the above-mentioned novel IL-15 mutation A body polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned plasmid, or the above-mentioned fusion body.
  • a detection kit comprising the above-mentioned novel IL-15 mutant polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned plasmid, or the above-mentioned fusion;
  • the above-mentioned detection kit is used for detecting pathogens and tumor cells.
  • the beneficial effects of the present invention are: the present invention innovatively develops a novel interleukin-15 mutant polypeptide that enhances the binding or biological activity of IL-15R ⁇ and ⁇ c receptors, and provides the Nucleic acid sequence and amino acid sequence of the novel interleukin 15 mutant polypeptide, and in some embodiments, vectors comprising these amino acids and host cells comprising these vectors are disclosed.
  • the invention also discloses a novel interleukin-15 mutant polypeptide pharmaceutical composition.
  • the antibodies and compositions disclosed here can be used in the diagnosis and treatment of diseases.
  • these novel interleukin-15 mutant polypeptides, nucleic acids, plasmids, and vectors are used to diagnose or treat autoimmune diseases or cancer and other related diseases.
  • FIG. 1a Novel interleukin-15 mutant polypeptides (WT-IL15, B1-A1, B1-H4, B1-H5, B2-C1, B2-G1, B2-H1, B3-A1, B4-B1, B4-B6) Reduction SDS-PAGE analysis of and stained with Coomassie brilliant blue;
  • Figure 1b Reduced SDS-PAGE analysis of novel interleukin-15 mutant polypeptides (B4-E1, R2-F7, R2-G7, R2-B9, R3-D1, R4-E5, R4-G8) stained with Coomassie brilliant blue ;
  • FIG. 2a shows the detection of the affinity of novel interleukin 15 mutant polypeptides (WT-IL15, B2-G1, B2-H1, B3-A1) to IL-15R ⁇ by biofilm interferometry (BLI);
  • Figure 2b shows the detection of affinity between novel interleukin 15 mutant polypeptides (B4-B1, B4-B6, B4-E1, R1-E5) and IL-15R ⁇ by biofilm interferometry (BLI);
  • Figure 3 Shows the evaluation of the biological activity of novel interleukin 15 mutant polypeptides by proliferation assays on CTLL-2.
  • N-terminal as used herein is also called amino-terminal, NH 2 -terminal, N-terminal or amine-terminal, is the beginning of a protein or polypeptide and refers to the free amine group (-NH 2 ) at the end of a polypeptide .
  • amino acid means one of the 20 naturally occurring amino acids or any non-natural analog thereof, which may be located at a specified position.
  • Protein herein means at least two covalently linked amino acids, which includes proteins, polypeptides, oligopeptides and peptides. Proteins may be composed of naturally occurring amino acids and peptide bonds, or of synthetic peptidomimetic structures, the peptidomimetics being "analogues”.
  • nucleic acid means a nucleic acid composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants and their synthetic non-naturally occurring analogs) linked by phosphodiester bonds. composed of polymers.
  • nucleotide polymers in which the nucleotides and linkages between them include non-naturally occurring synthetic analogs such as, but not limited to, phosphorothioate, phosphoroamidate, methylphosphonate, chiral Methyl phosphate, 2'-O-methyl ribonucleotide, peptide nucleic acid (PNA), etc.
  • these polynucleotides can be synthesized using an automatic DNA synthesizer.
  • oligonucleotide generally refers to short polynucleotides, usually no greater than about 50 nucleotides. It is to be understood that when a nucleotide sequence is represented by a DNA sequence (ie A, T, G, C), this also includes an RNA sequence (ie A, U, G, C) wherein "U" is substituted for "T".
  • nucleotide sequences the 5' end of the left-hand end of a single-stranded nucleotide sequence; the left-hand direction of a double-stranded nucleotide sequence is called the 5' direction.
  • the direction in which 5' to 3' nucleotides are added to the nascent RNA transcript is called the direction of transcription.
  • the strand of DNA that has the same sequence as mRNA is called the coding strand.
  • encoding means the inherent property of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA or mRNA, used to synthesize other polymers and macromolecules in a biological process with a defined nucleotide sequence.
  • a gene encodes a protein if the transcription and translation of the mRNA produced by that gene produces the protein in a cell or other biological system.
  • the coding strand (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a template for transcription, gene or cDNA) can be referred to as coding proteins. Or other products of the gene or cDNA.
  • a "nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate forms of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.
  • Plasmid as used herein means a plasmid artificially constructed on the basis of natural plasmids to adapt to laboratory operations.
  • a nucleic acid molecule can be introduced into a host cell, thereby producing a transformed host cell.
  • a vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication, and may also include one or more selectable marker genes and other genetic elements known in the art.
  • a "host cell” as used herein is also called a recipient cell, and refers to a host cell that receives a foreign gene in transformation and transduction (infection).
  • “Pharmaceutically acceptable carrier” as used herein means a conventional pharmaceutically acceptable carrier.
  • Remington's Pharmaceutical Sciences, EW Martin, Mack Publishing Co., Easton, Pa., 15th ed. (1975) describes compositions suitable for drug delivery of one or more therapeutic compounds or molecules (e.g., one or more antibodies) and preparations, and additional agents.
  • preventing means inhibiting the full development of the disease.
  • Treatment refers to therapeutic intervention to ameliorate the signs or symptoms of a disease or pathological condition after it has begun to develop.
  • an "effective prophylactic/therapeutic dose” herein means an amount of a particular agent sufficient to achieve the desired effect in a subject treated with the agent.
  • the precise dosage will depend on the purpose of the treatment and can be determined by those skilled in the art using well known techniques. Dosage ranges may be 0.01-100 mg/kg body weight or greater, eg 0.1, 1, 10 or 50 mg/kg body weight, preferably 1-10 mg/kg.
  • Dosage ranges may be 0.01-100 mg/kg body weight or greater, eg 0.1, 1, 10 or 50 mg/kg body weight, preferably 1-10 mg/kg.
  • autoimmune disease refers to a disease in which the immune system mounts an immune response (eg, a B-cell or T-cell response) against a portion of the normal host's antigens (ie, self-antigens) and subsequently damages tissues.
  • Autoantigens may originate from host cells, or may be derived from commensal organisms, such as microorganisms that normally colonize mucosal surfaces (termed commensal organisms).
  • Autoimmune diseases affecting mammals include but are not limited to rheumatoid arthritis, juvenile oligoarthritis, collagen-induced arthritis, adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis, experimental Autoimmune encephalomyelitis, inflammatory bowel disease (eg, Crohn's disease, ulcerative colitis), autoimmune gastric atrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes, non-obese diabetes, Myasthenia gravis, Graves' disease, Hashimoto's thyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemic lupus erythematosus, autoimmune thrombocytopenic purpura, Goodpasture syndrome, Addison's disease, systemic sclerosis , Polymyositis, dermatomyositis, autoimmune hemolytic anemia, pernicious anemia, etc.
  • the novel interleukin-15 mutant polypeptide of the present invention can be used to treat cancer.
  • the cancers described in the present invention include, but are not limited to, lymphoma, blastoma, sarcoma (including liposarcoma), neuroendocrine tumor, mesothelioma, schwannoma, meningioma, adenoma, melanoma, and non-leukemic leukemia or Lymphoid malignancies.
  • squamous cell carcinoma e.g., squamous cell carcinoma
  • lung cancer small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma, peritoneal carcinoma, hepatocellular carcinoma, gastric cancer , gastrointestinal cancer, pancreatic cancer, malignant glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer , kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penis cancer, testicular cancer, esophageal cancer, bile duct tumor, head cancer, neck cancer, bone marrow stromal tumor, osteoclastoma, multiple myeloma, Osteolytic bone cancers, central nervous system tumors, brain tumors (glioma, neuroblasto
  • the present invention discloses a novel IL-15 mutant polypeptide, the novel IL-15 mutant polypeptide comprises the amino acid sequence shown in SEQ ID NO: 1, wherein:
  • X1 is selected from any amino acid in A and N;
  • X2 is selected from any amino acid in S, D, Y;
  • X3 is selected from any amino acid in D and F;
  • X4 is selected from D, G, any amino acid in N;
  • X5 is selected from any amino acid in E and Y;
  • X6 is selected from any amino acid in I, Q, and K;
  • X7 is selected from any amino acid in N and Y;
  • X8 is selected from any amino acid in Q, S, F, A;
  • X9 is selected from any amino acid in M and H;
  • X10 is selected from any amino acid in I, A, H;
  • the invention also discloses a nucleic acid molecule encoding the above-mentioned novel IL-15 mutant polypeptide.
  • the invention also discloses a plasmid containing the above-mentioned nucleic acid molecule.
  • the invention also discloses a host cell containing the above plasmid.
  • the present invention also discloses a fusion body, which is connected by a fusion partner to the N-terminal and/or C-terminal of the novel IL-15 mutant polypeptide;
  • the fusion partner is at least one of heterologous proteins, adhesion molecules, nucleic acid molecules, small molecular compounds, toxins, immune cells, and detectable markers;
  • the heterologous protein is an antibody, an antigen, a cytokine, a soluble receptor domain, a ligand, an enzyme, a peptide or a protein domain; the antigen is derived from an adeno-associated virus;
  • the immune cells are chimeric antigen receptor T cells.
  • the present invention also discloses a pharmaceutical composition, which contains the above-mentioned novel IL-15 mutant polypeptide, or the above-mentioned fusion body, and a pharmaceutically acceptable carrier in an effective preventive or therapeutic dose.
  • the present invention also discloses the use of the above-mentioned novel IL-15 mutant polypeptide, or the above-mentioned pharmaceutical composition in the preparation of medicines for biological material treatment.
  • the present invention also discloses a method for treating autoimmune disease, inflammatory disease, neurodegenerative disease, cancer or pathogen infection, comprising administering to the subject an effective therapeutic amount of the above-mentioned novel IL-15 mutant polypeptide, The aforementioned nucleic acid molecule, the aforementioned plasmid, or the aforementioned fusion.
  • the invention also discloses a detection kit, which contains the above-mentioned novel IL-15 mutant polypeptide, the above-mentioned nucleic acid molecule, the above-mentioned plasmid, or the above-mentioned fusion body; and the above-mentioned detection kit is used for detecting pathogens and tumor cells.
  • the three receptors bind to three different directions of IL-15 respectively. Therefore, when modifying the interface residues of IL-15 and its receptor ⁇ , ⁇ , it will not affect the binding of IL-15 and receptor ⁇ .
  • the crystal structure to determine the key sites for the interaction of IL-15 and its receptor ⁇ as N4, S7, D8, D61, E64, N65, I68, N72, and the key sites for the interaction of IL-15 and its receptor ⁇
  • the loci are D30, H32, Q108, M109, I111, N112.
  • the positive cloned gene was cloned into the eukaryotic expression vector PTT, and the successfully constructed plasmid was extracted and then transiently co-transfected into Expi293 cells for expression, and purified by protein G resin (Ying Tianlei et al., JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287 (23): 19399-19408). The purity of the purified protein was verified by SDS-PAGE gel, and the results are shown in Figure 1.
  • Anti-human IgG Fc Capture (AHC) probe (purchased from GE) was used to detect the binding ability of novel interleukin-15 mutant polypeptides to IL-15R ⁇ .
  • the soluble expression preparation of the novel interleukin-15 mutant polypeptide was basically carried out according to the literature (Ying Tianlei et al., JOURNAL OF BIOLOGICAL CHEMISTRY, 2012, 287(23): 19399-19408). During the detection process, the novel interleukin-15 mutant polypeptide was diluted by 3 times (the detection of affinity with IL-15R ⁇ was at an initial concentration of 1000nM to 12.35nM dilution).
  • a 96-well culture plate (Costar, USA), starting from a concentration of 4 ⁇ g/mL in a volume of 50 ⁇ L of RPMI medium supplemented with 10% fetal bovine serum (FBS) and 50 ⁇ g/mL gentamicin, Perform B1-A1, B1-H4, B1-H5, B2-C1, B2-G1, B2-H1, B3-A1, B4-B1, B4-B6, B4-E1, R2-F7, R2-G7, R2 - Serial dilutions of B9, R3-D1, R4-E5, R4-G8, WT-15 and sFc.
  • CTLL-2 cells were previously washed 5 times with RPMI medium and added to the plate at 5 ⁇ 10 3 cells/well in a volume of 50 ⁇ L.

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Abstract

本申请提供了IL-15突变体多肽及其制备方法和应用。进一步提供了编码该IL-15突变体多肽的核酸、包含该核酸的载体和宿主细胞。

Description

一种新型白介素15突变体多肽的开发及其应用
相关申请的交叉引用
本申请要求于2021年05月28日提交中国专利局，申请号为2021105911186，发明名称为“一种新型白介素15突变体多肽的开发及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物技术领域，具体涉及一种新型白介素15突变体多肽、其制备方法、及构建该新型白介素15突变体多肽所需的核酸、质粒、宿主细胞，相应的药用组合物及其应用。
背景技术
白介素15(IL-15)是1994年Grabstein等在检测猴肾表皮细胞系CV21/EBNA上清液中发现的一种可溶性细胞因子，它可以维持CTLL-2细胞的增殖，因其与IL-2有许多相似的生物活性而备受关注。IL-2和IL-15都属于IL-2家族，IL-2家族成员受体都含γc链：包括IL-2、IL-4、IL-7、IL-9、IL-15和IL-21。其中，IL-2和IL-15具备共同的IL-2/IL-15Rβ(CD122)和γc受体(CD132)。IL-2的特异性受体为IL-2Rα(CD25)，主要表达在活化的T细胞表面，IL-2是促进T细胞增殖最重要的细胞因子；IL-15的特异性受体为IL15Rα(CD215)。
在自然情况下，IL-15可以结合到细胞表面的IL-15β/γ受体,并激活下游的STAT5信号通路，进而促进CD8+T细胞的分化和扩增。在体内，IL-2主要是游离状态存在，因此只要和细胞表面受体结合就可以发挥作用。而IL-15和IL-15Rα主要在DC细胞和单核细胞表面表达，游离状态较少。因此IL-15信号通路的活化更多的是在细胞和细胞接触之后才能激活下游信号通路发挥作用。IL-15信 号传递经由IL-15Rα、IL-15Rβ及γc的异三聚体复合物。IL-15作用机制为转递呈作用，一个细胞将其细胞膜结合的IL-15/IL-15Rα复合物递呈给另一个表达IL-15Rβ及γc受体的靶细胞，实际上是“细胞-细胞”间的相互作用，这种方式要求一个细胞必须与另一个细胞发生接触才能产生作用，这既是机体免疫系统调控的需要，也在一定程度上制约了利用IL-15/IL-15Rα复合物进行相关疾病的免疫治疗。
因此，利用合成生物学技术开发新型的IL-15/IL-15Rα复合物是一个具有重要意义的研究。本发明人在长效化蛋白领域创新性的利用合成生物学开发了基于抗体IgG Fc的新型单体Fc(CN109705211B；202011161007.3)，本申请的新型白介素15突变体多肽引入了此新型单体Fc。同时利用IL-15转递呈作用机制，创新性地开发了具有增强或降低IL-15Rβ及γc受体的结合或生物学活性的新型白介素15突变体多肽。
发明内容
本申请的主要目的在于提供一种新型IL-15突变体多肽及其应用。
为了实现上述目的，本发明提供如下技术方案：
一种新型IL-15突变体多肽，包含SEQ ID NO:1所示的氨基酸序列，其中：
X1选自A、N中的任一种氨基酸；
X2选自S、D，Y中的任一种氨基酸；
X3选自D、F中的任一种氨基酸；
X4选自D、G，N中的任一种氨基酸；
X5选自E、Y中的任一种氨基酸；
X6选自I、Q，K中的任一种氨基酸；
X7选自N、Y中的任一种氨基酸；
X8选自Q、S，F，A中的任一种氨基酸；
X9选自M、H中的任一种氨基酸；
X10选自I、A，H中的任一种氨基酸；
与天然IL-15氨基酸相比，对于IL-15Rβ/γ具有增加或减少的结合活性。
上述一种新型IL-15突变体多肽，作为一种优选的实施方案，
所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为A，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2选自D，Y中的任一氨基酸，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
更优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为F，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4选自G，N中的任一氨基酸，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
更进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为Y，X6为I，X7为N，X8为Q，X9为M，X10为I。
上述一种新型IL-15突变体多肽，作为一种优选的实施方案：所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6选自Q，K中的任一氨基酸，X7为N，X8为 Q，X9为M，X10为I；
优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为Y，X8为Q，X9为M，X10为I；
更优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为Y，X8选自S，F，A中的任一氨基酸，X9为M，X10为I；
进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为H，X10为I；
更进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10选自A，H中的任一氨基酸。
SEQ ID NO:1(IL-15突变体的氨基酸序列)
Figure PCTCN2022094443-appb-000001
SEQ ID NO:2(突变体B1-A1的氨基酸序列)
Figure PCTCN2022094443-appb-000002
SEQ ID NO:3(突变体B1-H4的氨基酸序列)
Figure PCTCN2022094443-appb-000003
Figure PCTCN2022094443-appb-000004
SEQ ID NO:4(突变体B1-H5的氨基酸序列)
Figure PCTCN2022094443-appb-000005
SEQ ID NO:5(突变体B2-C1的氨基酸序列)
Figure PCTCN2022094443-appb-000006
SEQ ID NO:6(突变体B2-G1的氨基酸序列)
Figure PCTCN2022094443-appb-000007
SEQ ID NO:7(突变体B2-H1的氨基酸序列)
Figure PCTCN2022094443-appb-000008
SEQ ID NO:8(突变体B3-A1的氨基酸序列)
Figure PCTCN2022094443-appb-000009
SEQ ID NO:9(突变体B4-B1的氨基酸序列)
Figure PCTCN2022094443-appb-000010
SEQ ID NO:10(突变体B4-B6的氨基酸序列)
Figure PCTCN2022094443-appb-000011
SEQ ID NO:11(突变体B4-E1的氨基酸序列)
Figure PCTCN2022094443-appb-000012
SEQ ID NO:12(突变体R2-F7的氨基酸序列)
Figure PCTCN2022094443-appb-000013
SEQ ID NO:13(突变体R2-G7的氨基酸序列)
Figure PCTCN2022094443-appb-000014
SEQ ID NO:14(突变体R2-B9的氨基酸序列)
Figure PCTCN2022094443-appb-000015
SEQ ID NO:15(突变体R3-D1的氨基酸序列)
Figure PCTCN2022094443-appb-000016
SEQ ID NO:16(突变体R4-E5的氨基酸序列)
Figure PCTCN2022094443-appb-000017
SEQ ID NO:17(突变体R4-G8的氨基酸序列)
Figure PCTCN2022094443-appb-000018
本发明的第二方面，提供一种核酸分子，编码上述的新型IL-15突变体多肽。
本发明的第三方面，提供一种质粒，所述的质粒含有上述的核酸分子。
本发明的第四方面，提供一种宿主细胞，所述的宿主细胞含有上述的质粒。
本发明的第五方面，提供一种融合体，所述融合体由融合伴侣与上述新型IL-15突变体多肽的N端和/或C端连接；
所述融合伴侣为异源蛋白质、粘附分子、核酸分子、小分子化合物、毒素、免疫细胞、可检测标记物中的至少一种；
所述异源蛋白质为抗体、抗原、细胞因子、可溶性受体结构域、配体、酶、肽或蛋白结构域；所述抗原来源于腺病毒相关病毒；
所述免疫细胞为嵌合抗原受体T细胞。
本发明的第六方面，提供一种药用组合物，所述的药用组合物含有有效预 防或治疗剂量的上述新型IL-15突变体多肽，或上述融合体，和一种药学可接受载体。
本发明的第七方面，提供一种上述IL-15突变体多肽、上述药用组合物在制备用于生物材料处理的药物中的用途。
本发明的第八方面，提供一种治疗自身免疫性疾病，炎症性疾病，神经退行性疾病，癌症或病原体感染的方法，包括向所述受试者施用有效治疗量的上述新型IL-15突变体多肽，上述核酸分子，上述质粒，或上述融合体。
本发明的第九方面，提供一种检测试剂盒，含有上述新型IL-15突变体多肽，上述核酸分子，上述质粒，或上述融合体；
上述检测试剂盒用于检测病原体、肿瘤细胞。
相对于现有技术，本发明的有益效果为：本发明创新性地开发了具有增强IL-15Rβ及γc受体的结合或生物学活性的新型白介素15突变体多肽，并提供了编码所述的新型白介素15突变体多肽的核酸序列及氨基酸序列，并在一些实施方案中公开了包含这些氨基酸的载体及包含这些载体的宿主细胞。
本发明还公开了新型白介素15突变体多肽药用组合物。这里公开的抗体和组合物可以用于疾病的诊断和治疗，在实施方案中，这些新型白介素15突变体多肽，核酸，质粒，和载体被用来诊断或治疗自身免疫疾病或癌症等相关疾病。
附图说明
图1a.新型白介素15突变体多肽(WT-IL15、B1-A1、B1-H4、B1-H5、B2-C1、B2-G1、B2-H1、B3-A1、B4-B1、B4-B6)的还原SDS-PAGE分析并用考马斯亮蓝染色图；
图1b.新型白介素15突变体多肽(B4-E1、R2-F7、R2-G7、R2-B9、R3-D1、R4-E5、R4-G8)的还原SDS-PAGE分析并用考马斯亮蓝染色图；
图2a.显示利用生物膜干涉技术(BLI)检测新型白介素15突变体多肽(WT-IL15、B2-G1、B2-H1、B3-A1)与IL-15Rβ的亲和力；
图2b.显示利用生物膜干涉技术(BLI)检测新型白介素15突变体多肽(B4-B1、B4-B6、B4-E1、R1-E5)与IL-15Rβ的亲和力；
图3.显示通过在CTLL-2上的增殖测定法来评价新型白介素15突变体多肽的生物学活性。
具体实施方式
为了使本技术领域的人员更好地理解本申请方案，下面将结合案例对本申请实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本申请一部分的实施例，而不是全部的实施例。基于本申请中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都应当属于本申请保护的范围。
为了能更彻底地理解发明，以下列出一些定义。上述定义意在包含语法等同成分。
本文中使用的“N端”也称为氨基末端，NH 2-末端，N-末端或胺末端，是蛋白质或多肽的起始，指的是位于多肽末端的游离胺基团(-NH 2)。“C端”也称为羧基末端，羧基末端，C-末端尾部，C-末端或COOH-末，是蛋白质或多肽的末端，由游离羧基(-COOH)末端终止。
本文中使用的“氨基酸”意指20种天然存在的氨基酸之一或任一非天然类似物，它们可位于具体规定的位置。
本文中“蛋白质”意指至少两个共价连接的氨基酸，其包括蛋白质、多肽、寡肽和肽。蛋白质可由天然存在的氨基酸和肽键构成，或由合成的肽模拟物结构构成，该肽模拟物即“类似物”。
本文所使用的“核酸”意指由核苷酸单元(核糖核苷酸，脱氧核糖核苷酸，相关的天然存在的结构变体及其合成的非天然存在的类似物)通过磷酸二酯键组成的聚合物。因此，该术语包括核苷酸聚合物，其中核苷酸和它们之间的键包括非天然存在的合成类似物，例如但不限于硫代磷酸酯，氨基磷酸酯，甲基磷酸酯，手性甲基磷酸酯，2'-O-甲基核糖核苷酸，肽核酸(PNA)等。例如，可以使用自动DNA合成仪合成这些多核苷酸。术语“寡核苷酸”通常是指短多核苷酸，通常不大于约50个核苷酸。应当理解，当核苷酸序列由DNA序列(即A，T，G，C)表示时，这也包括其中“U”取代“T”的RNA序列(即A，U，G，C)。
本文使用常规符号来描述核苷酸序列：单链核苷酸序列的左手末端5'末端；双链核苷酸序列的左手方向称5'方向。向新生RNA转录物添加5'至3'核苷酸的方向称为转录方向。具有与mRNA相同序列的DNA链被称为编码链。
本文中所使用的“编码”意指多核苷酸中特定核苷酸序列的固有特性，例如基因，cDNA或mRNA，用作在具有确定的核苷酸序列的生物过程中合成其他聚合物和大分子的模板，或确定的氨基酸序列和由此产生的生物学特性。因此，如果由该基因产生的mRNA的转录和翻译在细胞或其他生物系统中产生蛋白质，则基因编码蛋白质。编码链(其核苷酸序列与mRNA序列相同并且通常在序列表中提供)和非编码链(用作转录模板，基因或cDNA)可以被称为编码蛋白质。或该基因或cDNA的其他产物。除非另有说明，否则“编码氨基酸序列的核苷酸序列”包括彼此简并形式且编码相同氨基酸序列的所有核苷酸序列。编码蛋白质和RNA的核苷酸序列可包括内含子。
本文中使用的“质粒”意指在天然质粒的基础上为适应实验室操作而进行人工构建的质粒。可将核酸分子导入宿主细胞，从而产生转化的宿主细胞。载 体可包括允许其在宿主细胞中复制的核酸序列，例如复制起点，还可以包括一种或多种选择标记基因和本领域已知的其他遗传元件。
本文所使用的“宿主细胞”也称为受体细胞，是指在转化和转导(感染)中接受外源基因的宿主细胞。
本文中使用的“药学可接受载体”意指常规的药学上可接受的载体。Remington's Pharmaceutical Sciences，EWMartin，Mack Publishing Co.，Easton,Pa.，第15版(1975)，描述了适用于药物递送一种或多种治疗化合物或分子(例如一种或多种抗体)的组合物和制剂，以及另外的药剂。
本文中所使用的“预防”疾病是指抑制疾病的完全发展。“治疗”是指在其开始发展后改善疾病或病理状况的体征或症状的治疗性干预。
本文中“有效预防/治疗剂量”意指足以在用该药剂治疗的受试者中达到所需效果的一定量的特定药剂。精确的剂量将依赖于治疗的目的，并可为本领域技术人员通过使用公知技术所确定。剂量范围可为0.01-100mg/kg体重或更大，例如0.1、1、10或50mg/kg体重，优选1-10mg/kg。如本领域所公知，对于抗体或Fc融合体降解、全身性或局部性递药和新蛋白酶合成速率，以及年龄、体重、大致健康状况、性别、饮食、给药时间、药物相互作用以及病症的严重程度而言，调整可以是必需的，并可由本领域那些技术人员通过常规的实验方法来确定。
本文中“自身免疫性疾病”指一种疾病，其中免疫系统针对正常宿主的一部分抗原(即自身抗原)产生免疫应答(例如，B细胞或T细胞应答)，随后对组织造成损伤。自身抗原可以源自宿主细胞，或者可以衍生自共生生物，例如通常定殖于粘膜表面的微生物(称为共生生物)。影响哺乳动物的自身免疫疾病包括但不限于类风湿性关节炎，幼年型少关节炎，胶原诱导的关节炎，佐剂诱 导的关节炎，斯耶格伦综合征，多发性硬化症，实验性自身免疫性脑脊髓炎，炎性肠病(例如，克罗恩病，溃疡性结肠炎)，自身免疫性胃萎缩，寻常型天疱疮，牛皮癣，白癜风，1型糖尿病，非肥胖性糖尿病，重症肌无力，格雷夫斯病，桥本氏甲状腺炎，硬化性胆管炎，硬化性唾液腺炎，系统性红斑狼疮，自身免疫性血小板减少性紫癜，Goodpasture综合征，艾迪生病，系统性硬化症，多发性肌炎，皮肌炎，自身免疫性溶血性贫血，恶性贫血等。
优选的，本发明的新型白介素15突变体多肽可用于治疗癌症。本发明所述癌症包括但不限于淋巴瘤、胚细胞瘤、肉瘤(包括脂肉瘤)、神经内分泌肿瘤、间皮瘤、神经鞘瘤、脑膜瘤、腺瘤、黑素瘤以及非白血性白血病或淋巴恶性肿瘤。上述癌症更具体的实例包括鳞状细胞癌(如，鳞状上皮细胞癌)、肺癌、小细胞肺癌、非小细胞肺癌、肺腺癌以及肺鳞状细胞癌、腹膜癌、肝细胞癌、胃癌、胃肠癌、胰腺癌、恶性胶质瘤、宫颈癌、卵巢癌、肝癌、膀胱癌、肝细胞瘤、乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫内膜或子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、睾丸癌、食道癌、胆管肿瘤，头癌、颈癌、骨髓基质瘤、破骨细胞瘤、多发性骨髓瘤、溶骨性癌(osteolytic bone cancers)、中枢神经系统肿瘤、脑肿瘤(神经胶质瘤、成神经细胞瘤、星细胞瘤、成神经管细胞瘤、室管膜细胞瘤和视网膜成神经细胞瘤)、鼻咽癌、基底细胞癌、胆管癌、卡波氏肉瘤、原发性肝癌或子宫内膜癌、以及血管系统肿瘤(血管肉瘤和hemagiopericytoma)。
本发明公开了一种新型IL-15突变体多肽，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中：
X1选自A、N中的任一种氨基酸；
X2选自S、D，Y中的任一种氨基酸；
X3选自D、F中的任一种氨基酸；
X4选自D、G，N中的任一种氨基酸；
X5选自E、Y中的任一种氨基酸；
X6选自I、Q，K中的任一种氨基酸；
X7选自N、Y中的任一种氨基酸；
X8选自Q、S，F，A中的任一种氨基酸；
X9选自M、H中的任一种氨基酸；
X10选自I、A，H中的任一种氨基酸；
与天然IL-15氨基酸相比，对于IL-15Rβ/γ具有增加或减少的结合活性。
本发明还公开了一种核酸分子，编码上述新型IL-15突变体多肽。
本发明还公开了一种质粒，含有上述核酸分子。
本发明还公开了一种宿主细胞，含有上述质粒。
本发明还公开了一种融合体，由融合伴侣与上述新型IL-15突变体多肽的N端和/或C端连接；
所述融合伴侣为异源蛋白质、粘附分子、核酸分子、小分子化合物、毒素、免疫细胞、可检测标记物中的至少一种；
所述异源蛋白质为抗体、抗原、细胞因子、可溶性受体结构域、配体、酶、肽或蛋白结构域；所述抗原来源于腺病毒相关病毒；
所述免疫细胞为嵌合抗原受体T细胞。
本发明还公开了一种药用组合物，含有有效预防或治疗剂量的上述新型IL-15突变体多肽，或上述融合体，和一种药学可接受载体。
本发明还公开了上述新型IL-15突变体多肽，或上述药用组合物在制备用于生物材料处理的药物中的用途。
本发明还公开了一种治疗自身免疫性疾病，炎症性疾病，神经退行性疾病，癌症或病原体感染的方法，包括向所述受试者施用有效治疗量的上述新型IL-15突变体多肽，上述核酸分子，上述质粒，或上述融合体。
本发明还公开了一种检测试剂盒，含有上述新型IL-15突变体多肽，上述核酸分子，上述质粒，或上述融合体；且上述检测试剂盒用于检测病原体、肿瘤细胞。
实施例中使用的标准的重组DNA技术和分子克隆技术是本领域所熟知的(Ausubel，F.M等人，Current Protocols in Molecular Biology,Greene Publishing Assoc.和Wiley-Interscience出版)，适用于微生物生长的材料和方法是本领域熟知的。主要化学、生物试剂购自KAPA Biosystems，New England Biolabs，TransGen Biotech，Thermo Fisher Scientific，OMEGA bio-tek等。
下面结合具体实施例对本发明进行详细说明。
实施例1
新型白介素15突变文库设计与构建
根据IL-15与其受体α、β、γ的结晶复合物结构(PDB ID：4GS7)来看，三种受体分别结合在IL-15的三个不同方向。因此，当改造IL-15和其受体β、γ的界面残基时，将不会对IL-15与受体α的结合产生影响。
因此，我们利用晶体结构确定了IL-15与其受体β相互作用的关键位点为N4，S7，D8，D61,E64,N65,I68，N72，及IL-15与其受体γ相互作用的关键位点为D30，H32，Q108，M109，I111，N112。
选定关键作用位点后，我们设计利用简并氨基酸引物，在关键氨基酸位点进行随机定点突变，并将其构建至噬菌体表达载体pComb3x中。我们创新性地利用单克隆蛋白捕获ELISA，将IL-15Rβ及γc受体作为捕获抗原，选取关键位 点突变单克隆与其反应，初步判断这些新型IL-15突变体与受体β、γ的亲和，以野生型IL-15作为对照。并将阳性克隆基因克隆到真核表达载体PTT中，将构建成功的质粒大提后瞬时共转Expi293细胞进行表达，通过protein G树脂进行纯化(应天雷等，JOURNAL OF BIOLOGICAL CHEMISTRY,2012，287(23)：19399-19408)。纯化的蛋白通过SDS-PAGE胶进行纯度验证，结果如图1。
实施例2
利用生物膜干涉技术(BLI)检测新型IL-15突变体多肽与IL-15Rβ的结合能力
利用Anti-human IgG Fc Capture(AHC)探针(购自GE)检测新型白介素15突变体多肽分别与IL-15Rβ的结合能力。
其中新型白介素15突变体多肽的可溶性表达制备基本按文献进行(应天雷等，JOURNAL OF BIOLOGICAL CHEMISTRY,2012，287(23)：19399-19408)。检测过程中，新型白介素15突变体多肽按照3倍倍比稀释(检测与IL-15Rβ亲和力是起始浓度为1000nM到12.35nM稀释)。
如图2结果所示，因为与IL-15Rβ结合的关键氨基酸位点突变，新型白介素15多变体多肽与IL-15Rβ结合发生了提高或者降低。
实施例3
在CTLL-2细胞系中评价新型IL-15突变体多肽的生物学活性
为了评价新型IL-15突变体多肽的生物学活性，使用在CTLL-2细胞系上的增殖测定法。按照下面所描述的程序，采用用溴化3-(4，5-二甲基噻唑-2-基)-2，5-二苯基四唑鎓(MTT)进行的线粒体染色(MossmanT.J.Immunol.Methods.1983,65(1-2):55-63)，通过这些细胞的增殖的刺激来测量生物学活性。
在96-孔培养板(Costar,USA)中，在50μL的体积的补充有10％胎牛血清(FBS)和50μg/mL庆大霉素的RPMI培养基中，从4μg/mL的浓度开始，进行B1-A1、B1-H4、B1-H5、B2-C1、B2-G1、B2-H1、B3-A1、B4-B1、B4-B6、B4-E1、R2-F7、R2-G7、R2-B9、R3-D1、R4-E5、R4-G8、WT-15和sFc的系列稀释。事先将CTLL-2细胞用RPMI培养基洗涤5次，并且在50μL的体积中按照5×103个细胞/孔添加至平板。
在37℃、5％CO2和98％的相对湿度下温育72小时。通过用MTT进行染色来测定细胞生存力。sFc不是在生物学上有活性的，因为它不诱导CTLL-2细胞的增殖，这与B1-H4、B1-H5、B2-G1、B2-H1、B3-A1、B4-E1、R4-E5(它们以剂量依赖性方式刺激增殖)不同，结果如图3所示。
对所公开的实施例的上述说明，使本领域专业技术人员能够实现或使用本发明。以上所述仅是本发明的优选实施方式，应当指出，对于本领域的技术人员，在不脱离本发明原理的前提下，还可以做出若干改进和补充，这些改进和补充也应当视为本发明的保护范围。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的，本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下，在其它实施例中实现。因此，本发明将不会被限制于本文所示的这些实施例，而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。

Claims (11)

  1. 一种新型IL-15突变体多肽，其特征在于，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中：
    X1选自A、N中的任一种氨基酸；
    X2选自S、D，Y中的任一种氨基酸；
    X3选自D、F中的任一种氨基酸；
    X4选自D、G，N中的任一种氨基酸；
    X5选自E、Y中的任一种氨基酸；
    X6选自I、Q，K中的任一种氨基酸；
    X7选自N、Y中的任一种氨基酸；
    X8选自Q、S，F，A中的任一种氨基酸；
    X9选自M、H中的任一种氨基酸；
    X10选自I、A，H中的任一种氨基酸；
    与天然IL-15氨基酸相比，对于IL-15Rβ/γ具有增加或减少的结合活性。
  2. 根据权利要求1所述的新型IL-15突变体多肽，其特征在于，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为A，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
    优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2选自D，Y中的任一氨基酸，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
    更优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为F，X4为D，X5为E，X6为I，X7为N， X8为Q，X9为M，X10为I；
    进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4选自G，N中的任一氨基酸，X5为E，X6为I，X7为N，X8为Q，X9为M，X10为I；
    更进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为Y，X6为I，X7为N，X8为Q，X9为M，X10为I。
  3. 根据权利要求1所述的新型IL-15突变体多肽，其特征在于，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6选自Q，K中的任一氨基酸，X7为N，X8为Q，X9为M，X10为I；
    优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为Y，X8为Q，X9为M，X10为I；
    更优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为Y，X8选自S，F，A中的任一氨基酸，X9为M，X10为I；
    进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为H，X10为I；
    更进一步优选地，所述新型IL-15突变体多肽包含SEQ ID NO:1所示的氨基酸序列，其中，X1为N，X2为S，X3为D，X4为D，X5为E，X6为I，X7为N，X8为Q，X9为M，X10选自A，H中的任一氨基酸。
  4. 一种核酸分子，其特征在于，编码权利要求1-3任一所述的新型IL-15突变体多肽。
  5. 一种质粒，其特征在于，含有权利要求4所述的核酸分子。
  6. 一种宿主细胞，其特征在于，含有权利要求5所述的质粒。
  7. 一种融合体，其特征在于，所述融合体由融合伴侣与权利要求1-3任一项所述新型IL-15突变体多肽的N端和/或C端连接；
    所述融合伴侣为异源蛋白质、粘附分子、核酸分子、小分子化合物、毒素、免疫细胞、可检测标记物中的至少一种；
    所述异源蛋白质为抗体、抗原、细胞因子、可溶性受体结构域、配体、酶、肽或蛋白结构域；所述抗原来源于腺病毒相关病毒；
    所述免疫细胞为嵌合抗原受体T细胞。
  8. 一种药用组合物，其特征在于，含有有效预防或治疗剂量的权利要求1-3任一项所述的新型IL-15突变体多肽，或权利要求7所述的融合体，和一种药学可接受载体。
  9. 权利要求1-3之一所述的新型IL-15突变体多肽，或权利要求8所述的药用组合物在制备用于生物材料处理的药物中的用途。
  10. 一种治疗自身免疫性疾病，炎症性疾病，神经退行性疾病，癌症或病原体感染的方法，包括向所述受试者施用有效治疗量的权利要求1-3任一项所述的新型IL-15突变体多肽，权利要求4所述的核酸分子，权利要求5所述的质粒，或权利要求7所述的融合体。
  11. 一种检测试剂盒，其特征在于，含有权利要求1-3任一项所述的新型IL-15突变体多肽，权利要求4所述的核酸分子，权利要求5所述的质粒，或权利要求7所述的融合体；
    上述检测试剂盒用于检测病原体、肿瘤细胞。
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