WO2024037322A1 - 一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途 - Google Patents

一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途 Download PDF

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WO2024037322A1
WO2024037322A1 PCT/CN2023/110205 CN2023110205W WO2024037322A1 WO 2024037322 A1 WO2024037322 A1 WO 2024037322A1 CN 2023110205 W CN2023110205 W CN 2023110205W WO 2024037322 A1 WO2024037322 A1 WO 2024037322A1
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protein
seq
mutant
cells
amino acid
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French (fr)
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周海平
洪海燕
张雪亮
李重阳
谭晓璐
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海徕科(北京)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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • A61K38/2086—IL-13 to IL-16
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/65—Peptidic linkers, binders or spacers, e.g. peptidic enzyme-labile linkers
    • 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
    • A61P31/12—Antivirals
    • 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
    • A61P37/04—Immunostimulants
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7155—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/91—Cell lines ; Processes using cell lines

Definitions

  • the invention belongs to the technical field of medical preparations, and specifically relates to IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer and its use.
  • IL-15 Human interleukin-15
  • T cells B cells and NK cells
  • IL-15 can activate, maintain and expand CD8+ memory T cells without activating regulatory T lymphocytes (Tregs, which have immunosuppressive functions).
  • IL-2 is the first cytokine to be identified. It was originally discovered from the culture supernatant of activated human T cells and is a soluble factor that mediates T cell proliferation. IL-2 was also the first cytokine approved by the FDA for cancer treatment. Although mainly secreted by CD4+ and CD8+ T cells under antigen stimulation, activated dendritic cells (DC), mast cells and NKT cells also produce small amounts of IL-2.
  • IL-15 has some similar functions to IL-2, including stimulating the proliferation of activated T cells, generating cytotoxic effector T cells, and activating and maintaining NK cells. They also promote B cells to induce immunoglobulin synthesis and regulate lymphatic homeostasis. But unlike IL-2, IL-15mRNA is expressed in various tissues, including hematopoietic cells and non-hematopoietic cells, such as keratinocytes, nerve cells, stromal cells, fibroblasts, etc. But unlike the widespread IL-15 mRNA expression, mature IL-15 protein production is mainly restricted to DCs and monocytes/macrophages.
  • IL-15 has a chemotactic effect on T cells: circulating lymphocytes home to peripheral lymph nodes, inhibit lymphocyte apoptosis, promote the activation and proliferation of T cells, and induce the production of cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • IL-15 not only promotes the production of memory CD8+ T cells, but also plays a crucial role in maintaining the number of memory CD8+ T cells in the body.
  • NK cells IL-15 also plays an important role in their activation and proliferation. In mice overexpressing IL-15, the number of NK cells increased significantly and enhanced the immune response.
  • IL-15 also plays an important role in the functional maturation of DC cells and macrophages. In DC cells, IL-15 can promote DC cells to express costimulatory factors and IFN- ⁇ , and improve the ability of DC cells to activate CD8+ T cells and NK cells.
  • IL-2 and IL-15 are type I four-alpha helix bundle cytokines and belong to the gamma receptor cytokine family. This group of cytokines shares the same receptor subunit ⁇ c and exhibits pleiotropic effects in modulating innate and adaptive immune responses.
  • the receptors for IL-2 and IL-15 are heterotrimers, except for the cytokine receptor subunit ⁇ c (also known as IL-2R ⁇ or CD132); they also share a ⁇ subunit, here referred to as IL- 2/15R ⁇ (also known as CD122).
  • the third and unique receptor subunits of IL-2 and IL-15 are IL-2R ⁇ and IL-15R ⁇ respectively.
  • IL-2R ⁇ IL-2R ⁇
  • sIL-2R ⁇ soluble form
  • IL-15R ⁇ as a unique component of the IL-15 receptor complex, is mainly expressed on monocytes and dendritic cells. Different from other ⁇ c family cytokines (such as IL-2), IL-15 as a cytokine first binds to IL-15R ⁇ expressing cells, and then the IL-15/IL-15R ⁇ complex is presented to activated T cells or IL-2/15R ⁇ and ⁇ c on NK cells form a high-affinity immune synapse.
  • IL-15 as a cytokine first binds to IL-15R ⁇ expressing cells, and then the IL-15/IL-15R ⁇ complex is presented to activated T cells or IL-2/15R ⁇ and ⁇ c on NK cells form a high-affinity immune synapse.
  • IL-2/15R ⁇ Due to the shared receptor subunit (IL-2/15R ⁇ ), IL-2 and IL-15 trigger several similar downstream signaling pathways, including Janus kinases (JAKs) signal transducers and activators of transcription (STATs), JAK1 interacts with IL-2/15R ⁇ , and JAK3 interacts with ⁇ c.
  • JAKs Janus kinases
  • STATs Janus kinases
  • JAK1 interacts with IL-2/15R ⁇
  • JAK3 interacts with ⁇ c.
  • IL-2 and IL-15 also display distinct functions in the body, particularly in adaptive immune responses.
  • IL-2 is required for the development and maintenance of regulatory T cells (Tregs), and IL-2 is closely associated with activation-induced cell death (AICD).
  • AICD activation-induced cell death
  • IL-2 When IL-2 is used to treat tumors, it may cause capillary leak syndrome as a side effect.
  • IL-15 does not mediate AICD, but inhibits IL-2-induced AICD, and does not cause capillary leak syndrome side effects.
  • IL-15 is a major force supporting the persistence of natural killer (NK) cells and memory CD8+ T cells.
  • N-803 (Anktiva, ALT-803) is composed of an IL-15 mutant combined with the sushi region/IgG1Fc fusion protein of IL-15/IL-15R ⁇ . Compared with wild-type IL-15, N-803 has good pharmacokinetic properties, lasts longer in the body, and has stronger anti-tumor activity.
  • the technical problem to be solved by the present invention is how to provide better IL-15 drugs.
  • the present invention provides a heterodimeric protein, which includes protein a and protein b, where the protein a includes an IL-15 mutant and a first Fc mutant;
  • the protein b includes the IL-15R ⁇ subunit sushi domain and other partial fragments of the IL-15R ⁇ subunit and a second Fc mutant;
  • the first Fc mutant and the second Fc mutant are selected from Knob modified Fc or Hole modified Fc, and the modification type of Knob modification or Hole modification of the first Fc mutant and the second Fc mutant is different;
  • the IL-15 mutant has an amino acid sequence selected from positions 1-111 of SEQ ID No. 18, positions 1-111 of SEQ ID No. 15, positions 1-114 of SEQ ID No. 14, and SEQ ID No. 17 The polypeptide of any one of positions 1-114 of SEQ ID No. 16 and positions 1-114 of SEQ ID No. 13;
  • the IL-15R ⁇ subunit sushi domain and other partial fragments of the IL-15R ⁇ subunit are polypeptides whose amino acid sequence is SEQ ID No. 2.
  • the "sushi region" in the "IL-15R ⁇ subunit sushi domain and other partial fragments of IL-15R ⁇ subunit” described in the present invention mainly refers to the peptide segment whose amino acid sequence is SEQ ID No. 2 No. 1-65.
  • the "other partial fragments" in the "IL-15R ⁇ subunit sushi domain and other partial fragments of IL-15R ⁇ subunit” mentioned in the present invention mainly refer to the amino acid sequence of SEQ ID No. 2 No. 66-78 (DPALVHQRPAPPS) of peptides.
  • the protein a further includes a linker, which connects the IL-15 mutant and the first Fc mutant.
  • the linker can be (GGGGS)n, and n is a natural number from 0 to 4. In one embodiment of the present invention, n is 3.
  • the protein a includes: the IL-15 mutant, a linker connected to the C-terminal of the IL-15 mutant, and a first Fc mutant connected to the C-terminal of the linker;
  • the protein b includes: the IL-15R ⁇ subunit sushi domain and other partial fragments, and a second Fc mutant connected to the C-terminus of the IL-15R ⁇ subunit sushi domain and other partial fragments.
  • the first Fc mutant or the second Fc mutant may be the Fc of antibody IgG1, IgG2, IgG3 or IgG4 or a mutant thereof;
  • the first Fc mutant or the second Fc mutant is selected from Fc of IgG4 or a mutant thereof.
  • serine 228 in the hinge region can be mutated to proline to obtain a stable IgG4 Fc mutant.
  • the protein a and the protein b are formed by combining the Knob into hole of Fc.
  • the Fc domain of one chain contains T366W mutations
  • the corresponding domain of the other chain contains T366S, L368A, and Y407V mutations
  • the Fc domain of one chain contains T350V, L351Y, F405A, and Y407V mutations.
  • the corresponding domain of the other chain contains T350V, T360L, K392L and T394V mutations; or other forms of Knob into hole combinations.
  • the amino acid sequence of the knob-modified Fc (second Fc mutant) is SEQ ID No. 9, and its nucleotide sequence is SEQ ID No. 37; the hole-modified Fc The amino acid sequence of (the first Fc mutant) is SEQ ID No. 10, and its nucleotide sequence is SEQ ID No. 38.
  • the protein a can be any one of A1)-A7):
  • amino acid sequence is the protein of SEQ ID No. 18;
  • amino acid sequence is the protein of SEQ ID No. 15;
  • amino acid sequence is the protein of SEQ ID No. 14;
  • amino acid sequence is the protein of SEQ ID No. 17;
  • amino acid sequence is the protein of SEQ ID No. 16;
  • amino acid sequence is the protein of SEQ ID No. 13;
  • a fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of the protein described in any one of A1) to A6).
  • the protein b can be any one of A8)-A10):
  • amino acid sequence is the protein of SEQ ID No. 19;
  • amino acid sequence is the protein of SEQ ID No. 20;
  • a fusion protein obtained by connecting a protein tag to the N-terminus or/and C-terminus of the protein described in A8) or A9).
  • protein a in the heterodimeric protein is a protein whose amino acid sequence is SEQ ID No. 18, and protein b is a protein whose amino acid sequence is SEQ ID No. 19.
  • the present invention provides biological materials related to the above-mentioned heterodimeric proteins, and the biological materials are any of the following:
  • B2 An expression cassette containing the nucleic acid molecule described in B1);
  • B3 A recombinant vector containing the nucleic acid molecule described in B1) or the expression cassette described in B2);
  • B4 A recombinant microorganism containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • B5 An animal cell line containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • B6 A plant cell line containing the nucleic acid molecule described in B1) or containing the expression cassette described in B2) or containing the recombinant vector described in B3);
  • the present invention provides a product, the active ingredient of the product is the above-mentioned heterodimeric protein; the use of the product is any of the following C1)-C6):
  • NK cells Improve the killing ability of NK cells against tumor cells (such as K562 cells).
  • the present invention provides a pharmaceutical composition, which is composed of the above-mentioned heterodimeric protein and other drugs.
  • the other drugs may be at least one of the following drugs: immune checkpoint drugs, cell engager bispecific antibodies, and cell therapy products.
  • the present invention provides the application of the above-mentioned heterodimeric protein or the above-mentioned biological material or the above-mentioned product or the above-mentioned pharmaceutical composition in any one of the following D1)-D12):
  • NK cells Improve the killing ability of NK cells against tumor cells (such as K562 cells).
  • the T cells include CD8+ T cells and/or CD4+ T cells.
  • the body may be a mammalian body.
  • the mammals include humans and mice.
  • the present invention provides a method for treating diseases, which method includes the following steps: administering the above-mentioned heterodimeric protein or the above-mentioned biological material or the above-mentioned product or the above-mentioned pharmaceutical combination to the patient. substance so that the patient can be treated.
  • the diseases include infectious diseases, tumors, blood diseases, inflammatory diseases and autoimmune diseases.
  • infectious diseases include but are not limited to viral infections (such as smallpox virus infection, HIV infection, HBV infection, etc.), bacterial infections, and fungal infections.
  • the tumors include, but are not limited to, melanoma, colorectal cancer, skin cancer, lymphoma, renal cell carcinoma, liver cancer, lung cancer, gastric cancer, and breast cancer.
  • the hematological diseases include, but are not limited to, anemia, acute myeloid leukemia, myelodysplastic syndrome, and T-cell large granular lymphocytic leukemia.
  • the autoimmune diseases include, but are not limited to, multiple sclerosis, psoriasis, rheumatoid arthritis, gastritis, and mucositis.
  • the product may be a drug or preparation.
  • carrier materials may also be added.
  • the carrier materials include but are not limited to water-soluble carrier materials (such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.), poorly soluble carrier materials (such as ethyl cellulose, cholesterol stearate, etc.), enteric carriers Materials (such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.).
  • water-soluble carrier materials such as polyethylene glycol, polyvinylpyrrolidone, organic acids, etc.
  • poorly soluble carrier materials such as ethyl cellulose, cholesterol stearate, etc.
  • enteric carriers Materials such as cellulose acetate phthalate and carboxymethyl ethyl cellulose, etc.
  • These materials can be used to make a variety of dosage forms, including but not limited to tablets, capsules, dropping pills, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, Oral tablets, suppositories, freeze-dried powder injections, etc. It can be ordinary preparations, sustained-release preparations, controlled-release preparations and various particulate drug delivery systems.
  • carriers are, for example, diluents and absorbing agents such as starch, dextrin, calcium sulfate, lactose, glycerin, etc.
  • Alcohol sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, aluminum silicate, etc.; wetting agents and adhesives, such as water, glycerin, polyethylene glycol, ethanol, propanol, Starch slurry, dextrin, syrup, honey, glucose solution, arabic slurry, gelatin slurry, sodium carboxymethylcellulose, shellac, methylcellulose, potassium phosphate, polyvinylpyrrolidone, etc.; disintegrating agents, such as dry starch , alginate, agar powder, fucoidyl starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium lauryl sulfonate, methyl cellulose, ethyl cellulose etc.; disintegration inhibitors, such as sucrose, tristearin, cocoa butter, hydrogenated oil, etc.; absorption enhancers, such as quaternary ammonium salts, sodium
  • Tablets can also be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or bi-layer and multi-layer tablets.
  • a wide variety of carriers known in the art may be used. Examples of carriers are, for example, diluents and absorbents such as glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, kaolin, talc, etc.; binders such as gum arabic, tragacanth, gelatin, and ethanol.
  • agar powder such as honey, liquid sugar, rice cereal or batter, etc.
  • disintegrating agents such as agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose, ethyl cellulose, etc.
  • a wide variety of carriers known in the art may be used.
  • the carrier include polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, semi-synthetic glycerides, and the like.
  • diluents commonly used in this field can be used, for example, water, ethanol, polyethylene glycol, 1, 3-Propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, polyoxyethylene sorbitol fatty acid esters, etc.
  • an appropriate amount of sodium chloride, glucose or glycerin can be added to the injection preparation.
  • conventional co-solvents, buffers, pH adjusters, etc. can also be added.
  • colorants, preservatives, fragrances, flavoring agents, sweeteners or other materials can also be added to the pharmaceutical preparations.
  • the above dosage forms can be administered by injection, including subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection.
  • the present invention invented an innovative IL-15 mutant-Fc/IL-15R ⁇ -Fc heterodimer protein, which has more biological activity than existing similar products.
  • the dimeric protein HL-015-9 provided by the present invention.
  • This dimeric protein can well stimulate the proliferation of NK cells and T cells, and can be used alone or in combination with other drugs (such as immune checkpoint drugs, cell engager bispecific antibodies or cell therapy products) to achieve Better results.
  • the heterodimeric protein provided by the present invention can improve the proliferation activity of CTLL-2 and Mo7e cells.
  • the heterodimeric protein provided by the present invention can improve the proliferation activity of NK cells, CD8+T cells, and CD4+T cells.
  • the heterodimeric protein provided by the present invention can promote NK cells to kill K562.
  • the dimeric protein HL-015-9 provided by the present invention inhibits tumor growth better than ALT-803 at the same injection dose.
  • Figure 1 shows the effects of different molecules designed in the first round on Mo7e cell proliferation.
  • Figure 2 shows the effects of different molecules designed in the first round on CTLL-2 cell proliferation.
  • Figure 3 shows the effect of the second round of designed IL-15 mutant-Fc/IL-15Ralpha fragment-Fc on the proliferation of Mo7e cells.
  • Figure 4 shows the effect of the second round of designed IL-15 mutant-Fc/IL-15Ralpha fragment-Fc on CTLL-2 cell proliferation.
  • Figure 5 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on NK cell proliferation.
  • Figure 6 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on CD4+ T cell proliferation.
  • Figure 7 shows the effects of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on CD8+ T cell proliferation.
  • Figure 8 shows the stimulating effect of different IL-15 mutants-Fc/IL-15Ralpha fragment-Fc on NK cell killing.
  • Figure 9 shows the efficacy of HL-015-9 in tumor-bearing mouse models.
  • IL-15/ Dimeric protein form of IL-15Ra Different IL-15/ Dimeric protein form of IL-15Ra.
  • the signal peptide of mouse IL-2 is added (the amino acid sequence of the signal peptide is shown in SEQ ID No. 39, and its corresponding nucleotide sequence is shown in SEQ ID No. 40) and restriction enzyme cleavage sites, and different mammalian cell secretion expression plasmids of IL-15/IL-15Ra dimer proteins were constructed by GenScript Biotech through conventional gene synthesis and molecular construction technologies.
  • heterodimeric protein HL-015-2 as an example to illustrate the construction process of recombinant expression plasmid in eukaryotic cells:
  • the signal peptide with the nucleotide sequence SEQ ID No. 40 is added upstream of the DNA molecule with the nucleotide sequence SEQ ID No. 28 to obtain a new DNA molecule named 28'.
  • the recombinant expression plasmid expresses the protein whose amino acid sequence is SEQ ID No. 12 and the protein whose amino acid sequence is SEQ ID No. 19. The two proteins form the dimer HL-015-2.
  • the knob amino acid sequence information of the Fc fragment is shown in SEQ ID No. 9, and its core
  • the nucleotide sequence is SEQ ID No.37
  • the amino acid sequence information of hole is shown in SEQ ID No.10
  • its nucleotide sequence is SEQ ID No.38
  • the amino acid sequence of the heterodimeric protein HL-015-1 is SEQ No.11 and SEQ ID No.20
  • their nucleotide sequences are SEQ ID No.27 and SEQ ID No.36 respectively
  • the amino acid sequence of heterodimeric protein HL-015-2 is SEQ ID No.12 and SEQ ID No.
  • amino acid sequences of the heterodimeric protein HL-015-3 are SEQ ID No. 12 and SEQ ID No. 20, whose nucleotide sequences are SEQ ID No. 28 and SEQ ID No. 36 respectively.
  • the construction process of recombinant expression plasmids of HL-015-1 and HL-015-3 is the same as that of HL-015-2. The only difference lies in the corresponding replacement of the target coding gene.
  • ALT-803 molecule is named ALT-803 in the patent and is a dimer composed of proteins with amino acid sequences SEQ ID No.3 and SED ID No.4 body);
  • HR-1 the amino acid sequence is SEQ ID No. 5 and SEQ A dimer composed of the protein of ID No. 6;
  • HR-2 the amino acid sequence is SEQ ID No.7SEQ ID No.8 protein composed of dimers
  • the sequence of IL-15 in HL-015-1 (digits 1-114 of SEQ ID No. 11) is consistent with the sequence of IL-15 in HR-2 (digits 1-114 of SEQ ID No. 7) ;
  • the sequence of IL-15R ⁇ subunit in HL-015-1 (No. 1-78 of SEQ ID No. 20) is better than the sequence of IL-15R ⁇ in HR-2 (No. 1-74 of SEQ ID No. 8).
  • the IL-15R ⁇ in HL-015-2 has 13 more amino acids in the sushi region (positions 1-65 in SEQ ID No. 6) than the IL-15R ⁇ in ATL-803 (sequence: DPALVHQRPAPPS).
  • IL-15 in HL-015-3 adopts the N72D mutation
  • HL-015-2 and HL-15-03 HL-015-3
  • the recombinant expression plasmids pCGS3-015-1, pCGS3-015-2, pCGS3-015-3, pCGS3-ALT-803, and pCGS3-HR in Example 1 were transfected respectively.
  • -1 and pCGS3-HR-2 each recombinant expression plasmid was transfected into 200 mL, the plasmid dosage was 0.7 ⁇ g/mL, and the culture time was 10 days.
  • the cell density increased significantly in the early stages of culture, then slowly decreased, and the cell viability was relatively high. When samples were collected on the 10th day, the cell viability was still 80%.
  • Recombinant expression plasmid pCGS3-015-3 Recombinant expression plasmid pCGS3-015-3
  • ALT-803 Expressed by recombinant expression plasmid pCGS3-ALT-803
  • HR-1 Expressed by recombinant expression plasmid pCGS3-HR-1
  • HR-2 Expressed by recombinant expression plasmid pCGS3-ALT-803 Plasmid pCGS3-HR-2 expression).
  • Example 3 Heterodimeric protein affects the proliferation activity of CTLL-2 and Mo7e cells.
  • Cytokine growth-dependent CTLL2 cells (Suzhou Dingding Biopharmaceutical Co., Ltd., Cat. No. TCM-C724) were cultured in RPMI 1640 medium supplemented with 200 U/ml IL-2 and 10% fetal calf serum at 37°C and 5.0% CO2. At logarithmic phase, cells were harvested by centrifugation at 1000 rpm for 5 min and washed three times with phosphate-buffered saline.
  • the heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1 and HR-2 of Example 2 were respectively serially diluted to obtain heterodimeric proteins. solution, add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 11 concentrations.
  • the concentrations of heterodimeric proteins in these 11 treatment wells are 500ng/ml, 166.67ng/ml, 55.56ng/ml, and 18.52ng/ml respectively. , 6.17ng/ml, 2.06ng/ml, 0.69ng/ml, 0.23ng/ml, 0.076ng/ml, 0.025ng/ml, 0.0085ng/ml, and set a negative control adding 10 ⁇ l PBS to each well (i.e. 0ng/ ml), set multiple wells for each concentration, and after culturing for another 2 days, add CCK8, and culture for 2 days at 37°C and 5.0% CO 2 Hour. The plate was then read at 450nm and 630nm to check cell growth, and the experiment was repeated three times.
  • the test method for IL-15/IL-15Ra dimer protein to stimulate the proliferation of Mo7e cells is similar to that of CTLL-2.
  • Mo7e cells were added with 8ng/ml GM-CSF and Culture in RPMI 1640 medium with 10% fetal bovine serum to logarithmic phase. Cells were collected and washed, then resuspended in RPMI 1640 medium without GM-CSF and added to a 96-well plate at 20,000 cells per well.
  • heterodimeric proteins HL-015-1, HL-015-2, HL-015-3, ALT-803, HR-1 and HR-2 of Example 2 were serially diluted with PBS respectively.
  • a heterodimer protein solution was obtained, and 10 ⁇ l of heterodimer protein solution was added to each well.
  • Each heterodimeric protein is treated with 8 concentrations.
  • the concentrations of heterodimeric proteins in these 8 treatment wells are 100ng/ml, 33.33ng/ml, 11.11ng/ml, and 3.70ng/ml respectively. , 1.23ng/ml, 0.41ng/ml, 0.046ng/ml, 0.015ng/ml, and set a negative control (i.e.
  • HL-015-2 had the best cell proliferation ability whether it stimulated CTLL-2 or Mo7e (the results are shown in Figures 1 and 2, and the logarithmic values in Figures 1 and 2 are based on 10). HL-015-1 is superior to HR-2.
  • IL-15R ⁇ subunit in HL-015-1 has 4 more amino acids than the IL-15R ⁇ in HR-2.
  • IL-15R ⁇ in HL-015-02 also has 13 more amino acids (DPALVHQRPAPPS) in the sushi region (positions 1-65 of SEQ ID No. 6) than IL-15R ⁇ in ATL-803.
  • Example 2 Based on the experimental results of Example 2 and Example 3, it was decided to select the IL-15N72D mutation shown in SEQ ID No. 3 and the IL-15R ⁇ -Fc shown in SEQ ID No. 19 to continue the following experiments.
  • the synthetic nucleotide sequence is a DNA molecule of SEQ ID No. 34 and the nucleotide sequence is a DNA molecule of SEQ ID No. 35. Add the signal peptide with the nucleotide sequence SEQ ID No.40 upstream of the DNA molecule with the nucleotide sequence SEQ ID No.34 to obtain a new DNA molecule named 34’.
  • the recombinant expression plasmid pCGS3-015-9 contains the HL-015-9 gene (the DNA molecule whose nucleotide sequence is SEQ ID No.34 and the DNA molecule whose nucleotide sequence is SEQ ID No.35), and the expressed amino acid sequence is SEQ
  • the protein and amino acid sequence of ID No. 18 is the protein of SEQ ID No. 19, and the two proteins form the heterodimeric protein HL-015-9.
  • the construction method of recombinant expression plasmids from HL-015-4 to HL-015-8 is the same as HL-015-9. The only difference is that the HL-015-9 gene is replaced accordingly to obtain the HL-015-4 gene (
  • the recombinant expression plasmids pCGS3-HL-015-4 and HL-015-5 genes (the nucleotide sequences are SEQ ID No. 29 and SEQ ID No. 35 respectively) (the nucleotide sequences are SEQ ID No. 30 and SEQ ID No. 35 respectively) ID No. 35)
  • recombinant expression plasmid pCGS3-HL-015-5, HL-015-6 gene (nucleotide sequences are SEQ ID No.
  • recombinant expression plasmid pCGS3-HL- 015-6, HL-015-7 genes nucleotide sequences are SEQ ID No. 32 and SEQ ID No. 35 respectively
  • recombinant expression plasmids pCGS3-HL-015-7, HL-015-8 genes nucleoside The recombinant expression plasmid pCGS3-HL-015-8 (the acid sequences are SEQ ID No. 33 and SEQ ID No. 35) respectively.
  • pCGS3-HL-015-4 table The protein whose amino acid sequence is SEQ ID No. 13 and the protein whose amino acid sequence is SEQ ID No. 19 constitute the heterodimeric protein HL-015-4.
  • pCGS3-HL-015-5 expresses a protein whose amino acid sequence is SEQ ID No. 14 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-5.
  • pCGS3-HL-015-6 expresses a protein whose amino acid sequence is SEQ ID No. 15 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-6.
  • pCGS3-HL-015-7 expresses a protein whose amino acid sequence is SEQ ID No. 16 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-7.
  • pCGS3-HL-015-8 expresses a protein whose amino acid sequence is SEQ ID No. 17 and a protein whose amino acid sequence is SEQ ID No. 19.
  • the two proteins form the heterodimeric protein HL-015-8.
  • the recombinant expression plasmids pCGS3-HL-015-4 to pCGS3-HL-015-9 were expressed and protein purified according to the method in Example 2. Then the activity was measured according to the method of Example 3. The results are shown in Figure 3 and Figure 4 (the logarithmic values in Figure 3 and Figure 4 are based on 10). Among all mutants, HL-015-9 is more effective in stimulating CTLL-2 Proliferation or Mo7e has the strongest proliferative activity.
  • the heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, and HL-015 were serially diluted with PBS according to the different types of dimer proteins.
  • -7. Obtain heterodimeric protein solution from HL-015-8 and HL-015-9, and add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 8 concentrations. The concentrations of heterodimeric proteins in these 8 treatment wells are 166.67ng/ml, 55.56ng/ml, 18.52ng/ml, and 6.17ng/ml respectively.
  • heterodimeric proteins HL-015-4, HL-015-5, HL-015-6, and HL-015-7 were serially diluted with PBS according to the different types of dimer proteins. , HL-015-8 and HL-015-9 to obtain a heterodimeric protein solution, and add 10 ⁇ l of heterodimeric protein solution to each well. Each heterodimeric protein is treated with 8 concentrations. The concentrations of heterodimeric proteins in these 8 treatment wells are 100ng/ml, 33.33ng/ml, 11.11ng/ml, and 3.70ng/ml respectively.
  • Example 3 1.23ng/ml, 0.41ng/ml, 0.046ng/ml, 0.015ng/ml, the experiment is repeated three times, and a negative control (i.e. 0ng/ml) of 10 ⁇ l PBS is added to each well, and duplicate holes are set for each concentration. .
  • the remaining operations are the same as in Example 3.
  • Example 5 Heterogeneous dimer protein stimulates the proliferation of NK cells, CD8+T cells, and CD4+T cells.
  • NK cells CD56+, CD3-
  • CD8+ T cells CD3+, CD8+
  • CD4+ T cells CD3+, CD8+
  • isolation kits to isolate NK cells, CD8+ T cells, and CD4+ T cells from human peripheral blood cells, respectively.
  • cell The isolated cells were cultured in RPMI 1640 medium containing 10% fetal calf serum at 37°C and 5.0% CO2 overnight, and the cells were collected and stained with carboxyfluorescein diacetate succinimide ester (CFSE) at 37 Incubate at °C for 15 minutes, then centrifuge and wash, and resuspend in RPMI1640 medium containing 10% fetal calf serum.
  • CFSE carboxyfluorescein diacetate succinimide ester
  • HL-015-9 has the strongest stimulation activity of NK cells, CD4+T cells and CD8+T cells.
  • the experiment was divided into 3 groups according to different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng respectively.
  • /ml, 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml the experimental setting was repeated three times. And set up a negative control (i.e. 0ng/ml) with 10 ⁇ l PBS added to each well, and set up duplicate holes for each concentration.
  • the experiment was divided into three groups according to the different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng/ ml, 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml, 0ng/ml
  • the experimental setting was repeated three times. And set up a negative control (i.e. 0ng/ml) with 10 ⁇ l PBS added to each well, and set up duplicate holes for each concentration.
  • the experiment was divided into 3 groups according to different types of dimeric proteins.
  • concentrations of dimeric proteins ALT-803, HL-015-6 and HL-015-9 were 50ng respectively.
  • /nl 12.5ng/ml, 3.13ng/ml, 0.78ng/ml, 0.20ng/ml, 0.049ng/ml, 0.012ng/ml, 0.031ng/ml, 0ng/ml
  • the experimental setting was repeated 3 times. And set up a negative control with 10 ⁇ l of PBS added to each well (i.e. 0ng/ml), and set up duplicate holes for the negative control.
  • K562 cells (Wuhan Punosai, Cat. No. CL-0130) were cultured in RPMI 1640 medium containing 5% fetal calf serum at 37°C and 5.0% CO for 48 hours, then BATDA dye was added and incubated at 37°C for 25 minutes.
  • NK cells cultured with culture medium alone were used as a control.
  • Killing rate (%) (mean value of the experimental group - mean value of the blank control group) / (mean value of the maximum release group - mean value of the blank control group).
  • Killing rate (%) (mean value of the experimental group - mean value of the blank control group) / (mean value of the maximum release group - mean value of the blank control group).
  • the experimental results are shown in Figure 8.
  • Each IL-15/IL- All 15Ra complexes can significantly improve the killing effect of NK cells on K562, among which HL-015-9 has the best activity.
  • mice 7-9 weeks old, female, weight range 17-23g, purchased from Beijing Vitong Lihua Experimental Animal Technology Co., Ltd., animal batch number 110011211108488925
  • CT26 cells mouse colon cancer cell line, cultured in In RPMI1640 culture medium containing 10% fetal bovine serum (preserved by Crown Branch)
  • a mouse colon cancer subcutaneous transplantation tumor model was established.
  • the tumor volume reaches 60-100 cubic millimeters, the drug will be administered in random groups (the experiment is divided into 7 groups, with 7 tumor-bearing mice in each group).
  • the control group is the buffer group, and each mouse is injected intraperitoneally with buffer at a rate of 10 ⁇ l/g body weight;
  • the first group is ALT-803, 0.05 mg/kg group.
  • Each mouse is intraperitoneally injected with a solution of heterodimeric protein ALT-803 at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.05mg/kg body weight;
  • the second group is ALT-803, 0.15 mg/kg group.
  • Each mouse is injected intraperitoneally with a solution of heterodimeric protein ALT-803 at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.15mg/kg body weight;
  • the third group is ALT-803, 0.6 mg/kg group.
  • Each mouse is intraperitoneally injected with heterodimeric protein ALT-803 solution at 10 ⁇ l/g body weight to ensure the dosage of heterodimeric protein ALT-803. is 0.6mg/kg body weight;
  • the fourth group is HL-015-9, 0.05 mg/kg group.
  • Each mouse is intraperitoneally injected with heterodimeric protein HL-015-9 solution at 10 ⁇ l/g body weight to make heterodimeric protein HL-015
  • the dosage of -9 is 0.05mg/kg body weight;
  • the fifth group is HL-015-9, 0.15 mg/kg group.
  • Each mouse is injected intraperitoneally with a solution of heterodimeric protein HL-015-9 at 10 ⁇ l/g body weight to make the heterodimeric protein HL-015
  • the dosage of -9 is 0.15mg/kg body weight;
  • Group 6 is HL-015-9, 0.6 mg/kg group. Each mouse is intraperitoneally injected with heterodimeric protein HL-015-9 solution at 10 ⁇ l/g body weight to make heterodimeric protein HL-015 The dosage of -9 is 0.6 mg/kg body weight.
  • the invention provides an IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein.
  • the heterodimer protein includes protein a and protein b.
  • the protein a can be an amino acid.
  • the sequence is a protein whose amino acid sequence is SEQ ID No. 18, and the protein b can be a protein whose amino acid sequence is SEQ ID No. 19.
  • the IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein and related biological materials provided by the invention can effectively stimulate the proliferation of NK cells and T cells.
  • dimerization Body protein HL-015-9 inhibits tumor growth better than ALT-803 at the same injection dose. Therefore, the IL-15 mutant-Fc/IL-15R ⁇ subunit-Fc heterodimer protein provided by the present invention can be used to prepare drugs or preparations for treating tumors and/or viral infections.

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Abstract

本发明公开了IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途。所述异源二聚体蛋白包括蛋白a和蛋白b，所述蛋白a包括IL-15突变体和第一Fc突变体；所述蛋白b包括IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段和第二Fc突变体；所述第一Fc突变体和所述第二Fc突变体选自Knob修饰的Fc或Hole修饰的Fc，且两者修饰类型不同。通过实验证明：本发明提供的异源二聚体蛋白可有效的刺激NK细胞和T细胞的增殖，体内实验表明二聚体蛋白HL-015-9在同等注射剂量下，对肿瘤的增长抑制均优于ALT-803，可用于制备治疗肿瘤和/或病毒感染等疾病的药物或制剂。

Description

一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途 技术领域
本发明属于医用配制品技术领域，具体涉及IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途。
背景技术
人白细胞素IL-15(interleukin-15，IL-15)是一种多效性细胞因子，具有激活T细胞、B细胞和NK细胞，并可介导这些细胞的增殖和存活的功能。此外，IL-15能激活、维持和扩增CD8+记忆性T细胞，而不激活调节性T淋巴细胞(Tregs，具有免疫抑制功能)。
IL-2是第一个被鉴定的细胞因子，它最初是从活化的人类T细胞培养上清中发现的，是一种介导T细胞增殖的可溶性因子。IL-2也是FDA批准用于癌症治疗的第一种细胞因子。虽然在抗原刺激下主要由CD4+和CD8+T细胞分泌，但活化的树突状细胞(DC)、肥大细胞和NKT细胞也产生少量的IL-2。
IL-15与IL-2具有某些类似的功能，包括刺激活化的T细胞增殖、产生细胞毒性效应T细胞以及激活和维持NK细胞。它们还促进B细胞诱导免疫球蛋白合成和调节淋巴稳态。但与IL-2不同，IL-15mRNA在各种组织中均有表达，包括造血细胞和非造血细胞，如角质形成细胞、神经细胞、基质细胞和成纤维细胞等等。但与广泛的IL-15mRNA表达不同，成熟的IL-15蛋白产生主要局限于DC和单核细胞/巨噬细胞。
IL-15对T细胞具有化学趋化作用：循环的淋巴细胞归巢至外周淋巴结，抑制淋巴细胞发生凋亡，并促进T细胞的活化增殖，诱导产生细胞毒性T淋巴细胞(CTL)。对于CD8+T细胞，IL-15除了能够促进其记忆性CD8+T细胞的产生，而且在维持体内记忆性CD8+T细胞的数目上也起着至关重要的作用。对于NK细胞，IL-15在其的活化与增殖中也起着重要作用。在过表达IL-15的小鼠体内，NK细胞的数目则明显增加，并能增强免疫反应。此外，IL-15在DC细胞及巨噬细胞的功能成熟中也扮演重要角色。在DC细胞中，IL-15能够促进DC细胞表达共刺激因子及IFN-γ，提高DC细胞活化CD8+T细胞及NK细胞的能力.
IL-2和IL-15是I型四α螺旋束细胞因子，属于γ受体细胞因子家族。这组细胞因子共享相同的受体亚单位γc，并表现出调节先天性和适应性免疫应答的多效性作用。IL-2和IL-15的受体都是异源三聚体，除了细胞因子受体亚单位γc(也称为IL-2Rγ或CD132)外；它们也共享β亚单位，这里称为IL-2/15Rβ(也称为CD122)。IL-2和IL-15的第三个也是独特的受体亚单位分别是IL-2Rα和IL-15Rα。
IL-2的α、β和γ受体通常在一个细胞表面，除细胞表面表达外，IL-2Rα还以可溶性形式(sIL-2Rα)存在于某些疾病中，包括炎症性疾病、移植排斥反应和大多数恶性疾病。
IL-15Rα作为IL-15受体复合物的独特组分，主要在单核细胞和树突状细胞上表达。与其他γc家族细胞因子(如IL-2)不同的是，IL-15作为细胞因子首先与IL-15Rα表达细胞结合，然后，IL-15/IL-15Rα复合物递呈给激活的T细胞或NK细胞上的IL-2/15Rβ和γc，形成高亲和力免疫突触，由于共享受体亚单位(IL-2/15Rβγ)，IL-2和IL-15触发几个类似的下游信号通路，包括Janus激酶(JAKs)信号转导蛋白和转录激活蛋白(STATs)，JAK1与IL-2/15Rβ相互作用，JAK3与γc相互作用。
尽管有这些相似之处，IL-2和IL-15在体内也显示出不同的功能，特别是在适应性免疫反应中。例如，调节性T细胞(Treg)的发育和维持需要IL-2，而IL-2与活化诱导的细胞死亡(AICD)密切相关。IL-2治疗肿瘤时，可能会引起毛细血管渗漏综合症副反应。相比之下，IL-15不介导AICD，而是抑制IL-2诱导的AICD，也不会引起毛细血管渗漏综合症副反应。IL-15是支持自然杀伤(NK)细胞和记忆CD8+T细胞持久性的主要力量。
基于IL-15在体内扩增和激活效应淋巴细胞的能力，它的治疗潜力正在不断被开发。同时，在协同免疫增强的联合策略中IL-15的作用也越来越受到关注。N-803(Anktiva,ALT-803)由IL-15突变体与IL-15/IL-15Rα的sushi区/IgG1Fc融合蛋白结合而成。和野生型的IL-15相比，N-803具有良好的药代动力学特性，在体内中存在时间更长，抗肿瘤活性也更强。
2022年ASCO上，ImmunityBio公司发布该临床数据表明：N803联合卡介苗(BCG)在160例BCG无响应的非肌层浸润性膀胱癌(NMIBC)患者中，2年总生存率为99％。在原位癌患者队列中，具有71％的完全缓解率，中位持续响应时间24.1个月。在乳头状患者队列中，18个月时无病生存率为53％。在2年的随访中，超过90％的患者避免了膀胱切除手术。对于BCG无响应的NMIBC患者，N-803+BCG的疗效和安全性超过了其它现有治疗方案。因此开发更好的IL-15药物有积极的意义。
发明公开
本发明要解决的技术问题是如何提供更好的IL-15药物。
为解决上述技术问题，第一个方面，本发明提供了一种异源二聚体蛋白，其包括蛋白a和蛋白b，所述蛋白a包括IL-15突变体和第一Fc突变体；所述蛋白b包括IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段和第二Fc突变体；所述第一Fc突变体和所述第二Fc突变体选自Knob修饰的Fc或Hole修饰的Fc，且所述第一Fc突变体和所述第二Fc突变体的Knob修饰或Hole修饰的修饰类型不同；
所述IL-15突变体为氨基酸序列选自SEQ ID No.18第1-111位、SEQ ID No.15第1-111位、SEQ ID No.14第1-114位、SEQ ID No.17第1-114位、SEQ ID No.16第1-114位和SEQ ID No.13第1-114位中任一种的多肽；
所述IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段为氨基酸序列是SEQ ID No.2的多肽。
本发明中所述“IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段”中的“sushi区域”主要是指氨基酸序列是SEQ ID No.2第1-65位的肽段。
本发明中所述“IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段”中的“其他部分片段”主要是指氨基酸序列是SEQ ID No.2第66-78位(DPALVHQRPAPPS)的肽段。
进一步地，所述蛋白a还包括连接子，所述连接子连接所述IL-15突变体和所述第一Fc突变体，连接子可为(GGGGS)n，n是0到4的自然数。在本发明的一个实施例中，所述n为3。
更进一步地，所述蛋白a包括：所述IL-15突变体、与所述IL-15突变体的C端相连的连接子、与所述连接子的C端相连的第一Fc突变体；所述蛋白b包括：IL-15Rα亚基sushi结构域及其他部分片段、与所述IL-15Rα亚基sushi结构域及其他部分片段的C端相连的第二Fc突变体。
上述异源二聚体蛋白中，所述第一Fc突变体或第二Fc突变体可以是抗体IgG1、IgG2、IgG3或IgG4的Fc或其突变体；
进一步地，所述第一Fc突变体或所述第二Fc突变体选自IgG4的Fc或其突变体。
更进一步地，如果采用IgG4形式，其铰链区的第228位丝氨酸可突变成脯氨酸，用以得到稳定的IgG4Fc突变体。
上述异源二聚体蛋白中，所述蛋白a与所述蛋白b是通过Fc的Knob into hole组合而形成的。两条肽链中，其中一条链Fc的结构域含有T366W突变，对应的另一条链的结构域含有T366S、L368A和Y407V突变；或者其中一条链Fc的结构域含有T350V、L351Y、F405A和Y407V突变，对应的另一条链的结构域含有T350V、T360L、K392L和T394V突变；或者是其他形式的Knob into hole的组合。
在本发明的一个实施例中，所述knob修饰的Fc(第二Fc突变体)的氨基酸序列为SEQ ID No.9，其核苷酸序列为SEQ ID No.37；所述hole修饰的Fc(第一Fc突变体)的氨基酸序列为SEQ ID No.10，其核苷酸序列为SEQ ID No.38。
上述异源二聚体蛋白中，所述蛋白a可为A1)-A7)中任意一项：
A1)氨基酸序列是SEQ ID No.18的蛋白质；
A2)氨基酸序列是SEQ ID No.15的蛋白质；
A3)氨基酸序列是SEQ ID No.14的蛋白质；
A4)氨基酸序列是SEQ ID No.17的蛋白质；
A5)氨基酸序列是SEQ ID No.16的蛋白质；
A6)氨基酸序列是SEQ ID No.13的蛋白质；
A7)在A1)-A6)任一项所述蛋白质的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
所述蛋白b可为A8)-A10)中任意一项：
A8)氨基酸序列是SEQ ID No.19的蛋白质；
A9)氨基酸序列是SEQ ID No.20的蛋白质；
A10)在A8)或A9)所述蛋白质的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
优选地，所述异源二聚体蛋白中的蛋白a为氨基酸序列是SEQ ID No.18的蛋白质，蛋白b为氨基酸序列是SEQ ID No.19的蛋白质。
为解决上述技术问题，第二个方面，本发明提供了与上述异源二聚体蛋白相关的生物材料，所述生物材料为下述任一种：
B1)编码上述异源二聚体蛋白的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的动物细胞系；
B6)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的植物细胞系；
B7)产生上述异源二聚体蛋白的宿主细胞。
为解决上述技术问题，第三个方面，本发明提供了一种产品，所述产品的活性成分为上述异源二聚体蛋白；所述产品的用途为如下C1)-C6)任一种：
C1)预防和/或治疗疾病；
C2)激活机体的免疫系统；
C3)提高或增强机体的免疫功能；
C4)抑制肿瘤生长；
C5)提高NK细胞和/或T细胞增殖活性；
C6)提高NK细胞对肿瘤细胞(如K562细胞)的杀伤能力。
为解决上述技术问题，第四个方面，本发明提供了一种药物组合物，所述药物组合物由上述异源二聚体蛋白和其它药物组成。
进一步地，所述其它药物可为如下药物中的至少一种：免疫检查点类药物、细胞接合器类双特异性抗体、细胞治疗产品。
为解决上述技术问题，第五个方面，本发明提供了上述异源二聚体蛋白或上述生物材料或上述产品或上述药物组合物在如下D1)-D12)中任一种中的应用：
D1)制备预防和/或治疗疾病的产品；
D2)制备激活机体免疫系统的产品；
D3)制备提高或增强机体免疫功能的产品；
D4)制备抑制肿瘤生长的产品；
D5)制备提高NK细胞和/或T细胞增殖活性的产品；
D6)制备提高NK细胞对肿瘤细胞(如K562细胞)的杀伤能力的产品；
D7)预防和/或治疗疾病；
D8)激活动物的免疫系统；
D9)提高或增强机体免疫功能；
D10)抑制肿瘤生长；
D11)提高NK细胞和/或T细胞增殖活性；
D12)提高NK细胞对肿瘤细胞(如K562细胞)的杀伤能力。
上述任一产品或应用中，所述T细胞包括CD8+T细胞和/或CD4+T细胞。
上述任一产品或应用中，所述机体可为哺乳动物机体。所述哺乳动物包括人和小鼠。
为解决上述技术问题，第六个方面，本发明提供了一种治疗疾病的方法，所述方法包括如下步骤：向患者施用上述异源二聚体蛋白或上述生物材料或上述产品或上述药物组合物，使所述患者得到治疗。
以上任一所述产品或应用或方法中，所述疾病包括传染病、肿瘤、血液病、炎性疾病和自身免疫性疾病。
所述传染病包括但不限于病毒感染(如天花病毒感染、HIV感染、HBV感染等)、细菌感染、真菌感染。
所述肿瘤包括但不限于黑色素瘤、结直肠癌、皮肤癌、淋巴瘤、肾细胞癌、肝癌、肺癌、胃癌、乳腺癌。
所述血液病包括但不限于贫血、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。
所述自身免疫性疾病包括但不限于多发性硬化症、银屑病、风湿性关节炎、胃炎、黏膜炎。
以上任一所述产品或应用或方法中，所述产品可为药物或制剂。在实际应用中制备所述药物或制剂时，还可加入载体材料。
所述载体材料包括但不限于水溶性载体材料(如聚乙二醇、聚乙烯吡咯烷酮、有机酸等)、难溶性载体材料(如乙基纤维素、胆固醇硬脂酸酯等)、肠溶性载体材料(如醋酸纤维素酞酸酯和羧甲乙纤维素等)。
使用这些材料可以制成多种剂型，包括但不限于片剂、胶囊、滴丸、气雾剂、丸剂、粉剂、溶液剂、混悬剂、乳剂、颗粒剂、脂质体、透皮剂、口含片、栓剂、冻干粉针剂等。可以是普通制剂、缓释制剂、控释制剂及各种微粒给药系统。为了将单位给药剂型制成片剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如淀粉、糊精、硫酸钙、乳糖、甘 露醇、蔗糖、氯化钠、葡萄糖、尿素、碳酸钙、白陶土、微晶纤维素、硅酸铝等；湿润剂与粘合剂，如水、甘油、聚乙二醇、乙醇、丙醇、淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、紫胶、甲基纤维素、磷酸钾、聚乙烯吡咯烷酮等；崩解剂，例如干燥淀粉、海藻酸盐、琼脂粉、褐藻淀粉、碳酸氢钠与枸橼酸、碳酸钙、聚氧乙烯、山梨糖醇脂肪酸酯、十二烷基磺酸钠、甲基纤维素、乙基纤维素等；崩解抑制剂，例如蔗糖、三硬脂酸甘油酯、可可脂、氢化油等；吸收促进剂，例如季铵盐、十二烷基硫酸钠等；润滑剂，例如滑石粉、二氧化硅、玉米淀粉、硬脂酸盐、硼酸、液体石蜡、聚乙二醇等。还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。为了将单位给药剂型制成丸剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如稀释剂与吸收剂，如葡萄糖、乳糖、淀粉、可可脂、氢化植物油、聚乙烯吡咯烷酮、高岭土、滑石粉等；粘合剂如阿拉伯胶、黄蓍胶、明胶、乙醇、蜂蜜、液糖、米糊或面糊等；崩解剂，如琼脂粉、干燥淀粉、海藻酸盐、十二烷基磺酸钠、甲基纤维素、乙基纤维素等。为了将单位给药剂型制成栓剂，可以广泛使用本领域公知的各种载体。关于载体的例子是，例如聚乙二醇、卵磷脂、可可脂、高级醇、高级醇的酯、明胶、半合成甘油酯等。为了将单位给药剂型制成注射用制剂，如溶液剂、乳剂、冻干粉针剂和混悬剂，可以使用本领域常用的所有稀释剂，例如，水、乙醇、聚乙二醇、1,3-丙二醇、乙氧基化的异硬脂醇、多氧化的异硬脂醇、聚氧乙烯山梨醇脂肪酸酯等。另外，为了制备等渗注射液，可以向注射用制剂中添加适量的氯化钠、葡萄糖或甘油，此外，还可以添加常规的助溶剂、缓冲剂、pH调节剂等。此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂、甜味剂或其它材料。
使用上述剂型可以经注射给药，包括皮下注射、静脉注射、肌肉注射和腔内注射等。
本发明取得的有益技术效果如下：
1、本发明通过计算机辅助设计，并经过生物学活性筛选，发明了创新的IL-15突变体-Fc/IL-15Rα-Fc异源二聚体蛋白，比已有的类似产品生物学活性更好，例如本发明提供的二聚体蛋白HL-015-9。该二聚体蛋白能够很好的刺激NK细胞和T细胞的增殖，可用单独或者与其他药物(如免疫检查点类药物、细胞接合器类双特异性抗体或者细胞治疗产品)联合使用，从而达到更好的效果。
2、本发明提供的异源二聚体蛋白可提高CTLL-2和Mo7e细胞增殖活性。
3、本发明提供的异源二聚体蛋白可提高NK细胞、CD8+T细胞，CD4+T细胞增殖活性。
4、本发明提供的异源二聚体蛋白可促进NK细胞杀伤K562。
5、本发明提供的二聚体蛋白HL-015-9在同等注射剂量下，对肿瘤的增长抑制均优于ALT-803。
附图说明
图1为第一轮设计的不同分子对Mo7e细胞增殖的影响。
图2为第一轮设计的不同分子对CTLL-2细胞增殖的影响。
图3为第二轮设计的IL-15突变体-Fc/IL-15Ralpha片段-Fc对Mo7e细胞增殖的影响。
图4为第二轮设计的IL-15突变体-Fc/IL-15Ralpha片段-Fc对CTLL-2细胞增殖的影响。
图5为不同IL-15突变体-Fc/IL-15Ralpha片段-Fc对NK细胞增殖的影响。
图6为不同IL-15突变体-Fc/IL-15Ralpha片段-Fc对CD4+T细胞增殖的影响。
图7为不同IL-15突变体-Fc/IL-15Ralpha片段-Fc对CD8+T细胞增殖的影响。
图8为不同IL-15突变体-Fc/IL-15Ralpha片段-Fc对NK细胞杀伤作用的刺激作用。
图9为HL-015-9在荷瘤小鼠模型中的药效。
实施发明的最佳方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、不同异源二聚体蛋白的表达质粒构建
根据野生型IL-15的序列信息(SEQ ID No.1)和IL-15受体alpha亚基的sushi区结构域(SEQ ID No.2第1-65位)，设计出不同IL-15/IL-15Ra的二聚体蛋白形式。根据设计的不同L-15/IL-15Ra二聚体蛋白氨基酸序列信息，添加鼠的IL-2的信号肽(信号肽的氨基酸序列见SEQ ID No.39，其对应的核苷酸序列见SEQ ID No.40)和酶切位点，并由金斯瑞生物通过常规的基因合成和分子构建技术，构建了不同的IL-15/IL-15Ra二聚体蛋白哺乳动物细胞分泌表达质粒。
下面以异源二聚体蛋白HL-015-2为例，说明真核细胞重组表达质粒的构建过程：
金斯瑞合成核苷酸序列是SEQ ID No.28的DNA分子和核苷酸序列是SEQ ID No.35的DNA分子。在核苷酸序列是SEQ ID No.28的DNA分子上游添加核苷酸序列为SEQ ID No.40的信号肽得到新的DNA分子命名为28’。用DNA分子28’替换表达载体pCGS3(Merck公司)的HindIII和XhoI酶切识别位点之间的片段(HindIII和XhoI酶切识别位点之间的小片段)，保持表达载体pCGS3的其他核苷酸序列不变，得到的重组表达质粒经测序正确后命名为pCGS3-015-2a。在核苷酸序列是SEQ ID No.35的DNA分子上游添加核苷酸序列为SEQ ID No.40的信号肽得到新的DNA 分子命名为35’。用DNA分子35’替换重组表达质粒pCGS3-015-2a的BstBI和PacI的酶切识别位点之间的片段(BstBI和PacI的酶切识别位点之间的小片段)，保持重组质粒pCGS3-015-2a其他核苷酸序列不变，得到的重组表达质粒经测序正确后命名为pCGS3-015-2。重组表达质粒表达氨基酸序列是SEQ ID No.12的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成二聚体HL-015-2。
IL-15-Linker Fc和不同长度IL15Ra胞外区-有或无linker-Fc通过knob into hole构建不同的异源二聚体分子，Fc片段的knob氨基酸序列信息见SEQ ID No.9，其核苷酸序列为SEQ ID No.37，hole的氨基酸序列信息见SEQ ID No.10，其核苷酸序列为SEQ ID No.38；其中异源二聚体蛋白HL-015-1的氨基酸序列为SEQ No.11和SEQ ID No.20，其核苷酸序列分别为SEQ ID No.27和SEQ ID No.36；异源二聚体蛋白HL-015-2的氨基酸序列为SEQ ID No.12和SEQ ID No.19，其核苷酸序列为SEQ ID No.28和SEQ ID No.35；异源二聚体蛋白HL-015-3的氨基酸序列为SEQ ID No.12和SEQ ID No.20，其核苷酸序列分别为SEQ ID No.28和SEQ ID No.36。
HL-015-1和HL-015-3重组表达质粒的构建过程与HL-015-2相同，其区别仅在于将目的编码基因进行对应的替换。
另制备阳性参照如下：
1)从专利CN103370339A中获取ALT-803分子的编码基因(ALT-803分子在专利中的名称为ALT-803，由氨基酸序列为SEQ ID No.3和SED ID No.4的蛋白质组成的二聚体)；
2)参照文章Scientific Reports(2018)8:7675，将IL-15/IL-15Ra Sushi-Fc通过二硫键连接起来的P2239分子命名为HR-1(由氨基酸序列是SEQ ID No.5和SEQ ID No.6的蛋白质组成的二聚体)；
3)参照文献Scientific Reports(2018)8:7675，将IL-15-Fc和IL-15Ra-sushi结构域-Fc通过Knob into hole连接起来的HRP00018分子命名为HR-2(由氨基酸序列为SEQ ID No.7SEQ ID No.8的蛋白质组成的二聚体)；
HL-015-1中的IL-15的序列(SEQ ID No.11的第1-114位)与HR-2中的IL-15的序列(SEQ ID No.7的第1-114位)一致；HL-015-1中的IL-15Rα亚基的序列(SEQ ID No.20的第1-78位)比HR-2中的IL-15Rα的序列(SEQ ID No.8的第1-74)多了4个氨基酸(序列：PAPP)。
HL-015-2中的IL-15Rα比ATL-803中的IL-15Rα的sushi区域(SEQ ID No.6中的第1-65位)多了13个氨基酸(序列：DPALVHQRPAPPS)。
HL-015-3与HL-015-1的区别在于HL-015-3中的IL-15采用了N72D的突变；而HL-015-2与HL-15-03的区别在于HL-015-3中的IL-15Rα与Fc之间比HL-15-2的多了(G4S)3linker。
本发明第一轮构建的分子命名及其氨基酸和核苷酸序列如表1所示。
表1：第一轮构建的分子命名及其氨基酸和核苷酸序列

实施例2：蛋白质表达及纯化
按照Thermofisher公司的ExpiCHOTM Expression System Kit使用说明书，分别转染实施例1中的重组表达质粒pCGS3-015-1、pCGS3-015-2、pCGS3-015-3、pCGS3-ALT-803、pCGS3-HR-1和pCGS3-HR-2，每个重组表达质粒转染200mL，质粒用量为0.7μg/mL，培养时间为10天。细胞密度在培养初期明显增加，然后缓慢下降，细胞活率较高，到第10天收样时，细胞活率还有80％。
离心收集上清，抽滤后经过MabSelect SuRe LX(厂家Cytiva，货号17-5474-02)纯化(CIP缓冲液：0.1M NaOH，平衡缓冲液：20mM PB，0.15M NaCl，pH 7.0，淋洗缓冲液1：20mM PB，1M NaCl，pH 7.0，淋洗缓冲液2：50mM柠檬酸-柠檬酸钠，pH 5.5，洗脱缓冲液：50mM柠檬酸-柠檬酸钠，pH 3.5)；洗脱液收集浓缩后按照3％的量用Superdex 200(厂家Cytiva，货号V521276)上样纯化(平衡和洗脱液为20mmol/L PB，150mmol/L NaCl，pH7.0)，收集需要的峰，用SEC-HPLC检测纯度，并根据各蛋白的理论消光系数用紫外分光光度计确定蛋白质浓度。获得异源二聚体蛋白HL-015-1(由重组表达质粒pCGS3-015-1表达)、HL-015-2(由重组表达质粒pCGS3-015-2表达)、HL-015-3(由重组表达质粒pCGS3-015-3表达)、ALT-803(由重组表达质粒pCGS3-ALT-803表达)、HR-1(由重组表达质粒pCGS3-HR-1表达)和HR-2(由重组表达质粒pCGS3-HR-2表达)。
实施例3、异源二聚体蛋白影响CTLL-2和Mo7e细胞增殖活性测定
细胞因子生长依赖型CTLL2细胞(苏州顶点生物医药有限公司，货号TCM-C724)在添加200U/ml IL-2和10％胎牛血清的RPMI 1640培养基中在37℃、5.0％CO2下培养到对数期，1000rpm离心5分钟收获细胞，并用磷酸盐缓冲盐水洗涤三次。然后用含有10％胎牛血清(测定培养基)的RPMI 1640培养基中重悬细胞，按照5000个细胞每孔加到96孔细胞培养板上，培养4小时(细胞因子饥饿)后，用PBS分别梯度稀释的实施例2的异源二聚体蛋白HL-015-1、HL-015-2、HL-015-3、ALT-803、HR-1和HR-2得到异源二聚体蛋白溶液，每孔加入10μl异源二聚体蛋白溶液。每种异源二聚体蛋白设置11个浓度的处理，这11个处理孔中的异源二聚体蛋白的浓度分别为500ng/ml，166.67ng/ml，55.56ng/ml，18.52ng/ml，6.17ng/ml，2.06ng/ml，0.69ng/ml，0.23ng/ml，0.076ng/ml，0.025ng/ml，0.0085ng/ml，并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔，再培养2天后，添加CCK8，并在37℃、5.0％CO2下培养2 小时。然后在450nm和630nm处读取平板，检查细胞生长情况，实验设置3次重复。
IL-15/IL-15Ra二聚体蛋白刺激Mo7e细胞(浙江美森细胞科技有限公司，货号CTCC-001-0368)增殖的试验方法与CTLL-2类似，Mo7e细胞在添加8ng/ml GM-CSF和10％胎牛血清的RPMI 1640培养基中培养至对数期。收集并洗涤细胞，然后在不含GM-CSF的RPMI 1640培养基中重悬细胞，并按照每孔20000个细胞添加到96孔板中。培养4小时后，用PBS分别梯度稀释的实施例2的异源二聚体蛋白HL-015-1、HL-015-2、HL-015-3、ALT-803、HR-1和HR-2得到异源二聚体蛋白溶液，每孔加入10μl异源二聚体蛋白溶液。每种异源二聚体蛋白设置8个浓度的处理，这8个处理孔中的异源二聚体蛋白的浓度分别为100ng/ml，33.33ng/ml，11.11ng/ml，3.70ng/ml，1.23ng/ml，0.41ng/ml，0.046ng/ml，0.015ng/ml，并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔，并在37℃、5.0％CO2下培养4天。4天后，添加CCK8(20μl/孔)，并在37℃、5.0％CO2下培养2小时。然后在450nm和630nm处读数检测细胞生长情况，实验设置3次重复。结果显示HL-015-2无论是刺激CTLL-2还是刺激Mo7e的细胞增殖能力最优(结果见图1和图2，图1和图2中的对数值以10为底)。HL-015-1优于HR-2，它们主要区别在于HL-015-1中的IL-15Rα亚基比HR-2中的IL-15Rα多了4个氨基酸。HL-015-02中的IL-15Rα也比ATL-803中的IL-15Rα的sushi区域(SEQ ID No.6中的1-65位)多了13个氨基酸(DPALVHQRPAPPS)。
实施例4、IL-15突变体设计以及异源二聚体蛋白的获得以及活性比较
根据实施例2和实施例3的实验结果决定选用SEQ ID No.3所示的IL-15N72D突变以及SEQ ID No.19所示的IL-15Rα-Fc继续进行下述实验。
利用相关的结构软件，分析IL-15与IL-15Rα、IL-15Rβ/γ的结构找出可能影响IL-15的氨基酸位点，并且进行计算机建模与分子对接，挑选可能增强IL-15活性的氨基酸突变，合成相应的基因，筛选获得的氨基酸突变位点如表2所示。
表2：IL15的突变体及其氨基酸序列

合成核苷酸序列是SEQ ID No.34的DNA分子和核苷酸序列是SEQ ID No.35的DNA分子。在核苷酸序列是SEQ ID No.34的DNA分子上游添加核苷酸序列为SEQ ID No.40的信号肽得到新的DNA分子命名为34’。用DNA分子34’替换表达载体pCGS3(Merck公司)的HindIII和XhoI酶切识别位点之间的片段(HindIII和XhoI酶切识别位点之间的小片段)，保持表达载体pCGS3的其他核苷酸序列不变，得到的重组表达质粒经测序正确后命名为pCGS3-015-9a。在核苷酸序列是SEQ ID No.35的DNA分子上游添加核苷酸序列为SEQ ID No.40的信号肽得到新的DNA分子命名为35’。用DNA分子35’替换重组表达质粒pCGS3-015-9a的BstBI和PacI的酶切识别位点之间的片段(BstBI和PacI的酶切识别位点之间的小片段)，保持重组质粒pCGS3-015-9a其他核苷酸序列不变，得到的重组表达质粒经测序正确后命名为pCGS3-015-9。重组表达质粒pCGS3-015-9含有HL-015-9基因(核苷酸序列是SEQ ID No.34的DNA分子和核苷酸序列是SEQ ID No.35的DNA分子)，表达氨基酸序列是SEQ ID No.18的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-9。
HL-015-4至HL-015-8的重组表达质粒的构建方法同HL-015-9，其区别仅在于，将HL-015-9基因进行对应的替换，得到HL-015-4基因(核苷酸序列分别是SEQ ID No.29和SEQ ID No.35)的重组表达质粒pCGS3-HL-015-4、HL-015-5基因(核苷酸序列分别是SEQ ID No.30和SEQ ID No.35)的重组表达质粒pCGS3-HL-015-5、HL-015-6基因(核苷酸序列分别是SEQ ID No.31和SEQ ID No.35)的重组表达质粒pCGS3-HL-015-6、HL-015-7基因(核苷酸序列分别是SEQ ID No.32和SEQ ID No.35)的重组表达质粒pCGS3-HL-015-7、HL-015-8基因(核苷酸序列分别是SEQ ID No.33和SEQ ID No.35)的重组表达质粒pCGS3-HL-015-8。pCGS3-HL-015-4表 达氨基酸序列是SEQ ID No.13的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-4。pCGS3-HL-015-5表达氨基酸序列是SEQ ID No.14的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-5。pCGS3-HL-015-6表达氨基酸序列是SEQ ID No.15的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-6。pCGS3-HL-015-7表达氨基酸序列是SEQ ID No.16的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-7。pCGS3-HL-015-8表达氨基酸序列是SEQ ID No.17的蛋白质和氨基酸序列是SEQ ID No.19的蛋白质，两蛋白质组成异源二聚体蛋白HL-015-8。
本发明第二轮所构建的分子命名及其氨基酸和核苷酸序列如表3所示。
表3：第二轮构建的分子命名及其氨基酸序列和核苷酸序列
按照实施例2的方法对重组表达质粒pCGS3-HL-015-4至pCGS3-HL-015-9进行表达和蛋白纯化。然后按照实施例3的方法进行活性测定，结果见图3和图4(图3和图4中的对数值以10为底)，在所有突变体中，HL-015-9在刺激CTLL-2增殖或Mo7e增殖活性最强。
CTLL-2细胞增殖实验中，根据二聚体蛋白类型的不同，用PBS分别梯度稀释的异源二聚体蛋白HL-015-4、HL-015-5、HL-015-6、HL-015-7、HL-015-8和HL-015-9得到异源二聚体蛋白溶液，每孔加入10μl异源二聚体蛋白溶液。每种异源二聚体蛋白设置8个浓度的处理，这8个处理孔中的异源二聚体蛋白的浓度分别为166.67ng/ml，55.56ng/ml，18.52ng/ml，6.17ng/ml，2.06ng/ml，0.69ng/ml，0.23ng/ml，0.076ng/ml，实验设置3次重复，并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔。其余操作与实施例3相同。
Mo7e细胞增殖实验中，根据二聚体蛋白类型的不同，用PBS分别梯度稀释的异源二聚体蛋白HL-015-4、HL-015-5、HL-015-6、HL-015-7、HL-015-8和HL-015-9得到异源二聚体蛋白溶液，每孔加入10μl异源二聚体蛋白溶液。每种异源二聚体蛋白设置8个浓度的处理，这8个处理孔中的异源二聚体蛋白的浓度分别为100ng/ml，33.33ng/ml，11.11ng/ml，3.70ng/ml，1.23ng/ml，0.41ng/ml，0.046ng/ml，0.015ng/ml，实验设置3次重复，并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔。其余操作与实施例3相同。
实施例5、异源二聚体蛋白对NK细胞、CD8+T细胞，CD4+T细胞增殖刺激作用
分别用NK细胞(CD56+，CD3-)、CD8+T细胞(CD3+，CD8+)，CD4+T细胞(CD3+，CD8+)分离试剂盒从人外周血细胞中分离NK细胞、CD8+T细胞和CD4+T细胞。分离后的细胞在含有10％胎牛血清的RPMI 1640培养基中在37℃、5.0％CO2下培养过夜，收集细胞并用羧基荧光素二醋酸盐琥珀酰亚胺酯染色(CFSE)在37℃下孵育15分钟，然后离心洗涤，并用含有10％胎牛血清的RPMI1640培养基重悬，按照每孔10000个细胞加到预先加入PBS稀释后获得的不同浓度异源二聚体溶液的96孔细胞培养板上，异源二聚体溶液分别为ALT-803、HL-015-6和HL-015-9溶液。培养3天之后，通过流式细胞仪分析CFSC阳性的细胞数目，来判断不同的IL-15/IL-15Rα异源二聚体对不同细胞生长的影响，由图5、图6和图7可知，HL-015-9刺激NK细胞、CD4+T细胞和CD8+T细胞增殖活性最强。
NK细胞增殖实验中，根据二聚体蛋白类型的不同，实验共分为3组，PBS梯度稀释后二聚体蛋白ALT-803、HL-015-6和HL-015-9的浓度分别为50ng/ml，12.5ng/ml，3.13ng/ml，0.78ng/ml，0.20ng/ml，0.049ng/ml，0.012ng/ml，0.031ng/ml，实验设置3次重复。并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔。
CD4+细胞增殖实验中，根据二聚体蛋白类型的不同，实验共分为3组，PBS梯度稀释后二聚体蛋白ALT-803、HL-015-6和HL-015-9浓度分别为50ng/ml，12.5ng/ml，3.13ng/ml，0.78ng/ml，0.20ng/ml，0.049ng/ml，0.012ng/ml，0.031ng/ml，0ng/ml，实验设置3次重复。并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，每个浓度设置复孔。
CD8+细胞增殖实验中，根据二聚体蛋白类型的不同，实验共分为3组，PBS梯度稀释后二聚体蛋白ALT-803、HL-015-6和HL-015-9的浓度分别为50ng/nl，12.5ng/ml，3.13ng/ml，0.78ng/ml，0.20ng/ml，0.049ng/ml，0.012ng/ml，0.031ng/ml，0ng/ml，实验设置3次重复。并设置每孔加入10μl PBS的阴性对照(即0ng/ml)，阴性对照设置复孔。
实施例6、异源二聚体蛋白对NK细胞杀伤K562的影响
在含有5％胎牛血清的RPMI 1640培养基中在37℃、5.0％CO2下培养K562细胞(武汉普诺赛，货号CL-0130)48小时，然后加入BATDA染液在37℃孵育25分钟，离心用培养基洗涤3次，按照每孔5000个细胞将标记的K562细胞加入到96孔培养板中，按照靶细胞与效应细胞1:10的比例分别加入预先用培养基(含有5％胎牛血清的RPMI 1640培养基)与终浓度为100ng/ml的异源二聚体蛋白ALT-803、HR-1、HL-015-9、HL-015-6、HL-015-4共培养72小时的NK细胞，将靶细胞和效应细胞在37℃、5.0％CO2孵育4小时，后离心上清加入铕溶液，检测溶液的荧光值，以仅用培养基培养的NK细胞作为对照。同时设立空白对照 组和最大释放组(最大释放组是在空白对照组中加入终浓度为0.05％的Triton X，孵育后加铕检测)。按照如下公式计算杀伤率：杀伤率(％)＝(实验组均值-空白对照组均值)/(最大释放组均值-空白对照组均值)，实验结果见图8，每种IL-15/IL-15Ra复合物都能够大幅提高NK细胞对K562的杀伤作用，其中HL-015-9的活性最佳。
实施例7、异源二聚体蛋白在动物学体内药效学评价
BALB/c小鼠(7-9周龄，雌性，体重范围17-23g，购自北京维通利华实验动物技术有限公司，动物批号110011211108488925)皮下接种CT26细胞(鼠结肠癌细胞系，培养在含有10％胎牛血清的RPMI1640培养液中，由中美冠科保存)，建立鼠结肠癌皮下移植肿瘤模型。待肿瘤体积为60-100立方毫米时，随机分组给药(实验共分为7组，每组7只荷瘤小鼠)，给药第一天即为day1，给药周期为day1、day4、day8和day11，给药体积按照10μl/g体重，每周两次测量肿瘤体积，并按照如下公式计算肿瘤体积：肿瘤体积(mm3)＝1/2×(a×b2)(其中a表示长径，b表示短径)。
将异源二聚体蛋白ALT-803用buffer溶解，得到异源二聚体蛋白ALT-803溶液；将异源二聚体蛋白HL-015-9用buffer溶解，得到异源二聚体蛋白HL-015-9溶液；其中buffer为注射用0.9％生理盐水。
其中对照组为buffer组，每只小鼠按照10μl/g体重腹腔注射buffer；
第1组为ALT-803，0.05mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白ALT-803溶液，使异源二聚体蛋白ALT-803的给药剂量为0.05mg/kg体重；
第2组为ALT-803，0.15mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白ALT-803溶液，使异源二聚体蛋白ALT-803的给药剂量为0.15mg/kg体重；
第3组为ALT-803，0.6mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白ALT-803溶液，使异源二聚体蛋白ALT-803的给药剂量为0.6mg/kg体重；
第4组为HL-015-9，0.05mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白HL-015-9溶液，使异源二聚体蛋白HL-015-9的给药剂量为0.05mg/kg体重；
第5组为HL-015-9，0.15mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白HL-015-9溶液，使异源二聚体蛋白HL-015-9的给药剂量为0.15mg/kg体重；
第6组为HL-015-9，0.6mg/kg组，每只小鼠按照10μl/g体重腹腔注射异源二聚体蛋白HL-015-9溶液，使异源二聚体蛋白HL-015-9的给药剂量为0.6mg/kg体重。
结果如图9所示，结果显示，在同样浓度条件下，HL-015-9抑制模型小鼠 的肿瘤生长能力均优于对应浓度的ALT-803。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
工业应用
本发明提供了一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体蛋白，所述异源二聚体蛋白包括蛋白a和蛋白b，所述蛋白a可为氨基酸序列是氨基酸序列是SEQ ID No.18的蛋白质，所述蛋白b可为氨基酸序列是氨基酸序列是SEQ ID No.19的蛋白质。通过实验证明，本发明提供的IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体蛋白及相关生物材料可以有效的刺激NK细胞和T细胞的增殖，体内实验表明二聚体蛋白HL-015-9在同等注射剂量下，对肿瘤的增长抑制均优于ALT-803。因此，本发明提供的IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体蛋白可用于制备治疗肿瘤和/或病毒感染的药物或制剂。

Claims (18)

  1. 异源二聚体蛋白，其包括蛋白a和蛋白b，所述蛋白a包括IL-15突变体和第一Fc突变体；所述蛋白b包括IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段和第二Fc突变体；所述第一Fc突变体和所述第二Fc突变体选自Knob修饰的Fc或Hole修饰的Fc，且所述第一Fc突变体和所述第二Fc突变体的Knob修饰或Hole修饰的修饰类型不同；
    所述IL-15突变体为氨基酸序列选自SEQ ID No.18第1-111位、SEQ ID No.15第1-111位、SEQ ID No.14第1-114位、SEQ ID No.17第1-114位、SEQ ID No.16第1-114位和SEQ ID No.13第1-114位中任一种的多肽；
    所述IL-15Rα亚基sushi结构域及IL-15Rα亚基其他部分片段为氨基酸序列是SEQ ID No.2的多肽。
  2. 根据权利要求1所述的异源二聚体蛋白，其特征在于：所述蛋白a还包括连接子，所述连接子连接所述IL-15突变体和所述第一Fc突变体，所述连接子为(GGGGS)n，n是0到4的自然数。
  3. 根据权利要求2所述的异源二聚体蛋白，其特征在于：所述n为3。
  4. 根据权利要求1-3任一项所述的异源二聚体蛋白，其特征在于：所述蛋白a包括：所述IL-15突变体、与所述IL-15突变体的C端相连的连接子、与所述连接子的C端相连的第一Fc突变体；所述蛋白b包括：IL-15Rα亚基sushi结构域及其他部分片段、与所述IL-15Rα亚基sushi结构域及其他部分片段的C端相连的第二Fc突变体。
  5. 根据权利要求1-4任一项所述的异源二聚体蛋白，其特征在于：所述第一Fc突变体或所述第二Fc突变体为抗体IgG1、IgG2、IgG3或IgG4的Fc或其突变体。
  6. 根据权利要求1-5任一项所述的异源二聚体蛋白，其特征在于：所述第一Fc突变体或所述第二Fc突变体为抗体IgG4的Fc或其突变体。
  7. 根据权利要求1-6任一项所述的异源二聚体蛋白，其特征在于：所述Knob修饰的Fc的氨基酸序列为SEQ ID No.9。
  8. 根据权利要求1-7任一项所述的异源二聚体蛋白，其特征在于：所述Hole修饰的Fc的氨基酸序列为SEQ ID No.10。
  9. 根据权利要求1-8任一项所述的异源二聚体蛋白，其特征在于：所述蛋白a为A1)-A7)中任意一项：
    A1)氨基酸序列是SEQ ID No.18的蛋白质；
    A2)氨基酸序列是SEQ ID No.15的蛋白质；
    A3)氨基酸序列是SEQ ID No.14的蛋白质；
    A4)氨基酸序列是SEQ ID No.17的蛋白质；
    A5)氨基酸序列是SEQ ID No.16的蛋白质；
    A6)氨基酸序列是SEQ ID No.13的蛋白质；
    A7)在A1)-A6)任一项所述蛋白质的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
  10. 根据权利要求1-9任一项所述的异源二聚体蛋白，其特征在于：所述蛋白b为A8)-A10)中任意一项：
    A8)氨基酸序列是SEQ ID No.19的蛋白质；
    A9)氨基酸序列是SEQ ID No.20的蛋白质；
    A10)在A8)或A9)所述蛋白质的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
  11. 与权利要求1-10任一项所述的异源二聚体蛋白相关的生物材料，其特征在于：所述生物材料为下述任一种：
    B1)编码权利要求1-10任一项所述异源二聚体蛋白的核酸分子；
    B2)含有B1)所述核酸分子的表达盒；
    B3)含有B1)所述核酸分子或含有B2)所述表达盒的重组载体；
    B4)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的重组微生物；
    B5)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的动物细胞系；
    B6)含有B1)所述核酸分子或含有B2)所述表达盒或含有B3)所述重组载体的植物细胞系；
    B7)产生权利要求1-10任一项所述异源二聚体蛋白的宿主细胞。
  12. 一种产品，其活性成分为权利要求1-10任一项所述的异源二聚体蛋白；
    所述产品的用途为如下C1)-C6)任一种：
    C1)预防和/或治疗疾病；
    C2)激活机体的免疫系统；
    C3)提高或增强机体的免疫功能；
    C4)抑制肿瘤生长；
    C5)提高NK细胞和/或CD8+T细胞和/或CD4+T细胞增殖活性；
    C6)提高NK细胞对肿瘤细胞的杀伤能力。
  13. 一种药物组合物，其由权利要求1-10任一项所述的异源二聚体蛋白与其它药物组成。
  14. 根据权利要求13所述的药物组合物，其特征在于：所述其它药物可为如下药物中的至少一种：免疫检查点类药物、细胞接合器类双特异性抗体、细胞治疗产品。
  15. 权利要求1-10任一项所述的异源二聚体蛋白或权利要求11所述的生物材料或权利要求12所述的产品或权利要求13或14所述的药物组合物在如下D1)-D12)中任一种中的应用：
    D1)制备预防和/或治疗疾病的产品；
    D2)制备激活机体免疫系统的产品；
    D3)制备提高或增强机体免疫功能的产品；
    D4)制备抑制肿瘤生长的产品；
    D5)制备提高NK细胞和/或T细胞增殖活性的产品；
    D6)制备提高NK细胞对肿瘤细胞的杀伤能力的产品；
    D7)预防和/或治疗疾病；
    D8)激活动物的免疫系统；
    D9)提高或增强机体免疫功能；
    D10)抑制肿瘤生长；
    D11)提高NK细胞和/或T细胞增殖活性；
    D12)提高NK细胞对肿瘤细胞的杀伤能力。
  16. 根据权利要求15所述的方法，其特征在于：所述疾病包括传染病、肿瘤、血液病、炎性疾病和自身免疫性疾病。
  17. 一种治疗疾病的方法，包括如下步骤：向患者施用权利要求1-10任一项所述的异源二聚体蛋白或权利要求11所述的生物材料或权利要求12所述的产品或权利要求13或14所述的药物组合物，使所述患者得到治疗。
  18. 根据权利要求17所述的方法，其特征在于：所述疾病包括传染病、肿瘤、血液病、炎性疾病和自身免疫性疾病。
PCT/CN2023/110205 2022-08-15 2023-07-31 一种IL-15突变体-Fc/IL-15Rα亚基-Fc异源二聚体及其用途 WO2024037322A1 (zh)

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