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WO2022141406A1 - Réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine 10 et son application antitumorale - Google Patents

Réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine 10 et son application antitumorale Download PDF

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Publication number
WO2022141406A1
WO2022141406A1 PCT/CN2020/142167 CN2020142167W WO2022141406A1 WO 2022141406 A1 WO2022141406 A1 WO 2022141406A1 CN 2020142167 W CN2020142167 W CN 2020142167W WO 2022141406 A1 WO2022141406 A1 WO 2022141406A1
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Prior art keywords
interleukin
tumor
cancer
cells
effective amount
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PCT/CN2020/142167
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English (en)
Chinese (zh)
Inventor
刘陈立
陈茜
董宇轩
郭旋
胡勇
张昊
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深圳先进技术研究院
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Priority to PCT/CN2020/142167 priority Critical patent/WO2022141406A1/fr
Publication of WO2022141406A1 publication Critical patent/WO2022141406A1/fr

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    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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]

Definitions

  • the invention belongs to the field of medical biology, and in particular relates to an interleukin-10 chemically modified messenger ribonucleic acid and a preparation method and application thereof.
  • Cytokines are a class of small molecular polypeptide substances that are synthesized and secreted by activated immune cells or non-immune cells after stimulation and can act on themselves or other cells, and have biological activities such as mediating and regulating immune inflammatory responses. Cytokines generally modulate immune responses by regulating cell growth, differentiation, and effects by binding to the corresponding receptors. Cytokines such as interleukin 2 (IL2) and interferon (IFN) have been discovered for a long time, and they have been used in tumor therapy-related research, and have achieved certain results. However, the direct use of cytokines as a treatment method will produce some serious toxic and side effects, and there are also problems such as short half-life, poor stability, and strong immunogenicity, which affect their clinical application.
  • IL2 interleukin 2
  • IFN interferon
  • Transcription is a process that occurs in organisms all the time, and it is the first step from genetic information DNA to protein synthesis.
  • a system containing conditions such as ribonucleic acid (RNA) transcriptase, ribonucleotides (NTP), etc. is used, and deoxyribonucleic acid (DNA) is used as a template to imitate the in vivo transcription process to generate RNA, through such a technology Then it can control the gene (template) of transcription, the process of transcription and the use of post-transcriptional RNA.
  • RNA ribonucleic acid
  • NTP ribonucleotides
  • DNA deoxyribonucleic acid
  • mRNA messenger ribonucleic acid
  • the present invention discovered and verified for the first time that the cytokine interleukin-10 is the target of tumor, and the anti-tumor effect can be achieved by administering interleukin-10, which can activate the interleukin-10 receptor, in a subject.
  • the present invention further realizes the purpose of tumor treatment by constructing interleukin 10 to chemically modify messenger ribonucleic acid, so that it can stably and continuously express cytokine interleukin 10 in cells.
  • One aspect of the present invention provides the use of a reagent capable of providing a therapeutically effective amount of interleukin 10 in the preparation of a medicament for preventing or inhibiting tumors;
  • a second aspect of the present invention provides the use of an agent capable of providing a therapeutically effective amount of interleukin 10 in the manufacture of a medicament for activating an interleukin 10 receptor or a pathway downstream of an interleukin 10 receptor to inhibit tumors.
  • the third aspect of the present invention provides the use of an agent capable of providing a therapeutically effective amount of interleukin 10 in preventing or treating tumors.
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the group consisting of a therapeutically effective amount of interleukin 10, a drug capable of expressing interleukin 10 in a subject, and a drug capable of stimulating the production of interleukin 10 in a subject. drug;
  • the drug capable of expressing interleukin-10 in a subject is selected from DNA capable of encoding interleukin-10, RNA capable of encoding interleukin-10, or a vector comprising the above-mentioned coding sequence;
  • the vector is selected from Aqueous solutions, saline solutions, buffer solutions, glucose solutions, liposomes, cationic liposomes, cationic polymers, lysophospholipids, viral vectors, plasmid vectors, mRNA vectors, microcyclic vectors or chemically synthesized vectors;
  • the drug capable of stimulating the production of interleukin-10 in a subject is selected from the group consisting of inducing mononuclear macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, Drugs that synthesize and secrete interleukin-10 by any one or more of NK cells, mast cells, neutrophils, and eosinophils.
  • NK cells For example, lipopolysaccharide, catecholamines, CD36 and p38 mitogen-activated protein (MAP) kinases.
  • MAP mitogen-activated protein
  • the therapeutically effective amount of interleukin 10 is selected from long-circulating, active tumor-targeting and/or low-degraded interleukin-10; Circulating and/or actively targeting preparations are loaded with interleukin 10, and or by chemically modifying interleukin 10 to achieve long-term circulation and/or active targeting; the poorly degraded interleukin 10 is an inhibitor that inhibits interleukin 10, or the inhibitor In combination with interleukin 10.
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is an agent capable of providing interleukin 10 at the transcriptional concentration of IL-10 receptor in the subject;
  • the drug that expresses interleukin 10 in vivo is the drug that can express the interleukin 10 that activates the transcriptional concentration of interleukin 10 receptor in the subject;
  • the drug that can stimulate the production of interleukin 10 in the subject is the drug that can stimulate the endogenous secretion of interleukin in the subject, and
  • the concentration of secreted interleukin reaches that of the drug that activates the transcription of the interleukin 10 receptor.
  • the tumor is a solid tumor or a non-solid tumor, preferably the solid tumor is selected from bladder tumor, colorectal tumor, breast tumor, melanoma, testicular tumor, prostate tumor, kidney tumor, adrenal tumor, and parotid tumor , liver tumor, uterine tumor, brain tumor, ovarian tumor, small cell lung cancer, non-small cell lung cancer, head and neck cancer, Hodgkin lymphoma, esophagus tumor, gastric cancer, cervical cancer, pancreatic tumor, endometrial cancer, gastrointestinal Stromal tumor, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity and oropharyngeal cancer, thyroid cancer, small bowel cancer, osteosarcoma, glioma, multiple myeloma, skin cancer, gallbladder cancer, bile duct cancer, Retinoblastoma, fallopian tube cancer, peritoneal cancer, bone cancer, glioblastoma
  • the tumor is a tumor expressing IL10 receptor on the surface of immune cells at the tumor site, preferably, the immune cells are selected from monocytes, macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells, mast cells, neutrophils and eosinophils.
  • the immune cells are selected from monocytes, macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells, mast cells, neutrophils and eosinophils.
  • a fourth aspect of the present invention provides a chemically modified messenger RNA, the messenger RNA has an open reading frame encoding at least one interleukin 10 protein, and the messenger RNA has undergone at least one chemical modification.
  • the chemical modification is selected from the base modification of nucleotides as 5'methylcytosine, pseudouracil, N6-methyladenosine, inosine, 5 hydroxymethylcytosine , N1 methyladenosine, 2-thiouracil, 2-thiocytosine, 3-methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5-fluorocytosine, 5-chlorocytosine , 6-azocytosine, azacytosine, 5,6-dihydrocytosine, N4-ethylcytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil , at least one of 5-methyluracil, 5-methyl-2-thiouracil, 2-thiouracil and 4-thiouracil.
  • nucleotides as 5'methylcytosine, pseudouracil, N6-methyladenosine, inosine, 5 hydroxymethylcytosine , N1
  • the open reading frame further comprises a sequence encoding a signal peptide.
  • a fifth aspect of the present invention provides a composition comprising the aforementioned chemically modified messenger ribonucleic acid.
  • the composition further comprises at least one medium selected from aqueous solutions, saline solutions, buffer solutions, glucose solutions, liposomes, cationic liposomes, cationic polymers, Lysophospholipids, viral vectors, plasmid vectors, mRNA vectors, microcircles or chemically synthesized vectors.
  • at least one medium selected from aqueous solutions, saline solutions, buffer solutions, glucose solutions, liposomes, cationic liposomes, cationic polymers, Lysophospholipids, viral vectors, plasmid vectors, mRNA vectors, microcircles or chemically synthesized vectors.
  • a sixth aspect of the present invention provides an anti-tumor pharmaceutical composition, which comprises a therapeutically effective amount of the aforementioned chemically modified messenger ribonucleic acid or the aforementioned composition.
  • the tumor is a tumor that expresses the interleukin 10 receptor on the surface of immune cells at the tumor site, preferably, the immune cells are selected from monocytes, macrophages, T helper cells , dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells, mast cells, neutrophils and eosinophils.
  • the immune cells are selected from monocytes, macrophages, T helper cells , dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells, mast cells, neutrophils and eosinophils.
  • the tumor is selected from bladder tumor, colorectal tumor, breast tumor, melanoma, testicular tumor, prostate tumor, nephroma, adrenal tumor, parotid tumor, liver tumor, uterine tumor, and brain tumor , ovarian tumor, small cell lung cancer, non-small cell lung cancer, head and neck cancer, Hodgkin lymphoma, esophageal tumor, gastric cancer, cervical cancer, pancreatic tumor, endometrial cancer, gastrointestinal stromal tumor, nasal cavity and paranasal tumor Sinus, nasopharyngeal, oral and oropharyngeal, thyroid, small bowel, osteosarcoma, glioma, multiple myeloma, skin, gallbladder, bile duct, retinoblastoma, fallopian tube, peritoneum Cancer, bone cancer, glioblastoma; leukemia, chronic lymphocytic leukemia, chronic myeloid leuk
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the aforementioned chemically modified messenger RNA, the aforementioned composition or the aforementioned antitumor drug composition.
  • a seventh aspect of the present invention provides a method for treating or preventing tumors, the method comprising the use of an agent capable of providing a therapeutically effective amount of interleukin 10 to a subject;
  • the method of administration includes intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration;
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the group consisting of a therapeutically effective amount of interleukin 10, a drug capable of expressing interleukin 10 in a subject, and a drug capable of stimulating the production of interleukin 10 in a subject. drug;
  • the drug capable of expressing interleukin-10 in a subject is selected from DNA capable of encoding interleukin-10, RNA capable of encoding interleukin-10, or a vector comprising the above-mentioned coding sequence;
  • the vector is selected from Aqueous solutions, saline solutions, buffer solutions, glucose solutions, liposomes, cationic liposomes, cationic polymers, lysophospholipids, viral vectors, plasmid vectors, mRNA vectors, microcyclic vectors or chemically synthesized vectors;
  • the drug capable of stimulating the production of interleukin-10 in a subject is selected from the group consisting of inducing mononuclear macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, Drugs that synthesize and secrete interleukin-10 by any one or more of NK cells, mast cells, neutrophils, and eosinophils.
  • NK cells For example, lipopolysaccharide, catecholamines, CD36 and p38 mitogen-activated protein (MAP) kinases.
  • MAP mitogen-activated protein
  • interleukin-10 can be used to inhibit tumors with interleukin-10 receptors.
  • interleukin 10 as a cytokine, has a tumor-promoting effect, or that it can be used to indicate tumors, and can only be used as an auxiliary for tumor treatment.
  • the present invention overcomes the technical prejudice and discovers for the first time that interleukin 10 has the effect of inhibiting tumor.
  • the present invention finds for the first time that with the occurrence and development of tumors, the concentration of interleukin 10 at different time points in or near the tumor varies greatly, showing a trend of first rising and then falling.
  • the decrease in the concentration at the later stage indicates that the tumor-induced endogenous IL-10 cannot achieve the anti-tumor effect, so the tumor volume increases with the decrease of the IL-10 concentration, which can only be achieved with a sufficient concentration of exogenously administered IL-10.
  • the present invention verifies that the anti-tumor effect can be achieved as long as a sufficient concentration of interleukin 10 is administered by constructing a messenger RNA chemically modified by interleukin 10.
  • the results of the chemically modified messenger ribonucleic acid of interleukin 10 of the present invention also verify that interleukin 10 is the target of tumor action, and as long as a sufficient concentration of interleukin 10 is administered, the effect of inhibiting tumors can be achieved.
  • the present invention greatly prolongs the half-life of interleukin-10 by constructing a messenger RNA chemically modified by interleukin-10.
  • the messenger ribonucleic acid of the present invention does not need a transcription link and has a quick effect.
  • the chemically modified messenger RNA of the present invention has less immunoreactivity and is more suitable for human or animal administration.
  • FIG. 1 is the change curve of IL10 concentration in tumor tissue at different time points in the mouse bladder cancer subcutaneous tumor model in Example 1.
  • FIG. 1 is the change curve of IL10 concentration in tumor tissue at different time points in the mouse bladder cancer subcutaneous tumor model in Example 1.
  • FIG. 2 is the change curve of tumor weight at different time points in the mouse bladder cancer subcutaneous tumor model in Example 1.
  • FIG. 3 is the change curve of tumor volume of mice receiving injection of IL10 antibody and not injected with IL10 antibody in Example 2.
  • FIG. 3 is the change curve of tumor volume of mice receiving injection of IL10 antibody and not injected with IL10 antibody in Example 2.
  • Figure 4 is the change curve of IL10 receptor mRNA corresponding to the addition of different concentrations of IL10 to RAW 264.7 cells cultured in vitro in Example 3.
  • FIG. 5 is the change curve of IL10 receptor mRNA corresponding to the addition of different concentrations of IL10 to Jurkat cells cultured in vitro in Example 3.
  • FIG. 5 is the change curve of IL10 receptor mRNA corresponding to the addition of different concentrations of IL10 to Jurkat cells cultured in vitro in Example 3.
  • Fig. 6 is the gel electrophoresis result of in vitro synthesis of IL10 cmRNA in Example 4.
  • Figure 7 shows the expression of IL10 after transfecting 1 ⁇ g of IL10 cmRNA to MB49 cells and RAW 264.7 cells respectively in Example 5. Compared with the control group of cells without IL10 cmRNA transfection, the expression of IL10 in the supernatant of both cell cultures was at a relatively high level, indicating that IL10 was normally translated and secreted into the extracellular space.
  • Figure 8 shows the changes in tumor size after intratumoral injection of 10 ⁇ g/40 ⁇ g IL10 cmRNA in the mouse subcutaneous bladder cancer model in Example 6.
  • the tumor growth of mice in the experimental group injected with IL10 cmRNA was significantly inhibited, proving that IL10 cmRNA has the therapeutic effect of inhibiting tumor growth.
  • Figure 9 shows the changes in the body weight of mice after intratumoral injection of 10 ⁇ g/40 ⁇ g IL10 cmRNA in the mouse subcutaneous bladder cancer model in Example 7. Compared with the control group without IL10 cmRNA injection, the body weight of mice in the experimental group injected with IL10 cmRNA did not decrease significantly after injection, which proves the safety of IL10 cmRNA.
  • a “therapeutically effective amount” means that an active ingredient of the present invention, when used alone or in combination with another therapeutic agent, protects a subject from tumorigenesis, slows tumor progression, reduces tumor complications, reduces tumor severity, reduces tumor recurrence, Any amount that reduces tumor duration, etc.
  • the ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to skilled practitioners, such as physicians, researchers, etc., such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, Alternatively, the assessment can be performed by measuring the activity of the agent in an in vitro assay.
  • Subject includes any human or non-human animal.
  • Non-human animals include, but are not limited to, vertebrates such as non-human primates, sheep, dogs, cats, pigs, rabbits, and rodents (eg, mice, rats, and guinea pigs).
  • tumor inhibiting refers to reversing, alleviating, ameliorating, inhibiting, slowing the development of a tumor or preventing tumor symptoms, complications or reducing the onset, progression, progression, severity or recurrence of a tumor Or biochemical markers associated with disease.
  • Some embodiments of the present invention provide the use of an agent capable of providing a therapeutically effective amount of interleukin 10 in the preparation of a medicament for preventing or inhibiting tumors.
  • Some embodiments of the present invention provide the use of an agent capable of providing a therapeutically effective amount of interleukin 10 in the manufacture of a medicament for activating a pathway downstream of interleukin 10 to inhibit tumors.
  • Some embodiments of the present invention provide the use of an agent capable of providing a therapeutically effective amount of interleukin 10 in preventing or treating tumors.
  • Yet another embodiment of the present invention provides a method of treating or preventing a tumor comprising the use of an agent capable of providing a therapeutically effective amount of interleukin 10 to a subject.
  • Yet another embodiment of the present invention provides the use of interleukin 10 as a tumor-inhibiting target.
  • the therapeutically effective amount refers to the dose of the agent for interleukin 10 capable of activating transcription of the interleukin 10 receptor in a subject.
  • the therapeutically effective amount refers to the dose of the agent for interleukin 10 capable of activating the mRNA of the interleukin 10 receptor in a subject to activate downstream pathways.
  • the therapeutically effective amount is selected from, for example, the concentration of interleukin 10 at the tumor site reaches 0.1 ng/mL or more, 0.2 ng/mL or more, 0.3 ng/mL or more, 0.4 ng/mL or more, 0.5 ng/mL or more ng/mL or more, 0.6ng/mL or more, 0.7ng/mL, more than 0.8ng/mL, more than 0.9ng/mL, or more than 1ng/mL. Or any range between 0.1ng/mL-10ng/mL.
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is an agent capable of providing a transcriptional concentration of interleukin 10 that activates the interleukin 10 receptor in a subject.
  • the drug capable of expressing interleukin 10 in a subject is a drug capable of expressing IL-10 at a transcriptional concentration of activating interleukin 10 receptors in a subject;
  • the drug capable of stimulating the production of interleukin 10 in a subject is a drug capable of stimulating The test subjects endogenously secrete interleukin, and the concentration of the secreted interleukin reaches the concentration of the drug that activates the transcription of the interleukin 10 receptor.
  • the agent for providing a therapeutically effective amount of interleukin 10 is the agent capable of providing a therapeutically effective amount of interleukin 10, which is capable of activating the interleukin 10 receptor on the surface of immune cells in a subject to achieve tumor killing by immune cells of reagents.
  • the immune cells are selected from mononuclear macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells, mast cells, neutrophils and eosinophils.
  • the above methods of administration include intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion).
  • an agent capable of providing a therapeutically effective amount of interleukin 10 is applied directly within or near the tumor.
  • an agent capable of providing a therapeutically effective amount of interleukin 10 is targeted for delivery to the tumor site by a targeted formulation.
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the group consisting of a therapeutically effective amount of interleukin 10, a drug capable of expressing interleukin 10 in a subject, and a drug capable of stimulating the production of interleukin 10 in a subject medicine.
  • the therapeutically effective amount of interleukin 10 is achieved by increasing the dose of the effective amount of interleukin 10 administered, or by decreasing the breakdown or metabolism of interleukin 10.
  • the method for reducing the decomposition or metabolism of interleukin 10 can be achieved by conventional preparation methods or chemical modifications in the art, for example, by increasing targeting, accelerating the entry of interleukin 10 into the action area as soon as possible, and reducing the decomposition or metabolism in the body circulation. It can also be achieved by inhibiting the action of an inhibitor of interleukin 10, such as inhibiting the action of an interleukin 10 antibody. Degradation can also be reduced by formulation.
  • the drug capable of expressing interleukin 10 in a subject is selected from DNA capable of encoding interleukin 10, RNA capable of encoding interleukin 10, or a vector comprising the above-mentioned coding sequence; the vector is selected from an aqueous solution , saline solution, buffer solution, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector, microcyclic vector or chemically synthesized vector.
  • the RNA is selected from mRNA.
  • the drug capable of stimulating the production of interleukin-10 in a subject is selected from the group consisting of inducing monocyte macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells
  • a drug that synthesizes and secretes interleukin 10 by any one or more of cells, mast cells, neutrophils, and eosinophils For example, lipopolysaccharide, catecholamines, CD36 and p38 mitogen-activated protein (MAP) kinases.
  • MAP mitogen-activated protein
  • the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the chemically modified messenger RNA described in the present invention or the composition described in the present invention.
  • an agent capable of providing a therapeutically effective amount of interleukin 10 as an active ingredient in some embodiments of the present invention, an agent capable of providing a therapeutically effective amount of interleukin 10 as an active ingredient.
  • an agent capable of providing a therapeutically effective amount of interleukin 10 as the sole active ingredient is provided.
  • the tumor is a tumor expressing IL10 receptor on the surface of immune cells, preferably bladder tumor, colorectal tumor, breast tumor, melanoma, testicular tumor, prostate tumor, kidney tumor, adrenal tumor , parotid tumor, liver tumor, uterine tumor, brain tumor, ovarian tumor, small cell lung cancer, non-small cell lung cancer, head and neck cancer, Hodgkin lymphoma, esophagus tumor, gastric cancer, cervical cancer, pancreatic tumor, endometrial cancer , Gastrointestinal stromal tumor, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, oral cavity and oropharyngeal cancer, thyroid cancer, small bowel cancer, osteosarcoma, glioma, multiple myeloma, skin cancer, gallbladder cancer, Cholangiocarcinoma, retinoblastoma, fallopian tube cancer, peritoneal cancer, bone cancer, glioblasto
  • Some specific embodiments of the present invention provide a chemically modified messenger RNA, the messenger RNA has an open reading frame encoding at least one interleukin 10 protein, and the messenger RNA has undergone at least one chemical modification.
  • the open reading frame is codon-optimized.
  • the interleukin-10 is human-derived interleukin-10 and mouse-derived interleukin-10.
  • the interleukin 10 has homology with sequences NC_000001.11, NM_010548.270%-100%, preferably 95%-100%, or 99%-100% homology sex.
  • the chemical modification is selected from the group consisting of nucleotide base modification 5'methylcytosine, pseudouracil, N6-methyladenosine, inosine, 5 hydroxymethylcytosine Pyrimidine or N1 methyladenosine.
  • 60%-100% of the uracil in the open reading frame is chemically modified, preferably 90%-100% of the uracil is chemically modified, more preferably 95%-100% of uracils are chemically modified.
  • the chemical modification of uracil is pseudouracil.
  • 60%-100% of the cytosines in the open reading frame are chemically modified, preferably 90%-100% of the cytosines are chemically modified, more preferably 95% %-100% of cytosines are chemically modified.
  • the chemical modification of the cytosine is 5' methylcytosine.
  • 100% of the cytosines in the open reading frame are chemically modified to 5' methylcytosine; 100% of the uracils are chemically modified to pseudouracil.
  • the open reading frame has upstream and or downstream non-coding sequences that enhance the expression of the open reading frame.
  • the upstream of the 5'-end non-coding region sequence further includes a 5'-end cap structure; the downstream of the 3'-end non-coding region sequence includes a polyadenylation sequence.
  • the open reading frame further comprises a sequence encoding a signal peptide.
  • compositions comprising the above chemically modified messenger ribonucleic acid.
  • the carrier further comprises at least one medium selected from the group consisting of aqueous solutions, saline solutions, buffer solutions, glucose solutions, liposomes, cationic liposomes, cationic polymers, Lysophospholipids, viral vectors, plasmid vectors, mRNA vectors, microcircles or chemically synthesized vectors.
  • the cationic polymer is selected from the group consisting of Dendrimers and Polyethylenimine (PEI).
  • an anti-tumor drug composition the anti-tumor drug combination comprises a therapeutically effective amount of interleukin-10, a drug capable of expressing interleukin-10 in a subject, and capable of stimulating the production of interleukin-10 in a subject medicine.
  • the therapeutically effective amount of interleukin 10 is achieved by increasing the dose of the effective amount of interleukin 10 administered, or by decreasing the breakdown or metabolism of interleukin 10.
  • the method for reducing the decomposition or metabolism of interleukin 10 can be achieved by conventional preparation methods in the art, for example, by increasing targeting, accelerating the entry of interleukin 10 into the action area as soon as possible, and reducing the decomposition or metabolism in the body circulation. It can also be achieved by inhibiting the action of an inhibitor of interleukin 10, such as inhibiting the action of an interleukin 10 antibody. Degradation can also be reduced by formulation.
  • the drug capable of expressing interleukin 10 in a subject is selected from DNA capable of encoding interleukin 10, RNA capable of encoding interleukin 10, and a vector comprising the above-mentioned coding sequence; the vector is selected from an aqueous solution , saline solution, buffer solution, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector or chemically synthesized vector.
  • the drug capable of stimulating the production of interleukin-10 in a subject is selected from the group consisting of inducing monocyte macrophages, T helper cells, dendritic cells, B cells, cytotoxic T cells, ⁇ T cells, NK cells
  • a drug that synthesizes and secretes interleukin 10 by any one or more of cells, mast cells, neutrophils, and eosinophils For example, lipopolysaccharide, catecholamines, CD36 and p38 mitogen-activated protein (MAP) kinases.
  • MAP mitogen-activated protein
  • the drug capable of expressing interleukin 10 in a subject is selected from the above chemically modified messenger RNA or the above vector.
  • the above-mentioned antitumor pharmaceutical composition further includes any one of pharmaceutical excipients.
  • the above-mentioned antitumor pharmaceutical composition further includes at least one other active ingredient for treating tumors.
  • the present invention also provides some examples, which disclose the preparation method of the chemically modified messenger ribonucleic acid according to the present invention, and the preparation method comprises the following steps:
  • RNA is synthesized through an in vitro transcription reaction, and during the in vitro transcription reaction, nucleotides with chemically modified bases are used to replace ordinary base nucleotides.
  • linear DNA is used as a template during the in vitro transcription reaction, and the linear DNA is DNA encoding any IL10.
  • adenine ribonucleotides 5' methylcytosine adenine ribonucleotides, pseudouracil adenine ribonucleotides, and anti-reverse caps are added during the in vitro transcription reaction. and guanine ribonucleotides.
  • IL10 cmRNA cytokine interleukin 10 messenger ribonucleic acid
  • a plasmid vector for in vitro transcription of cytokines was constructed.
  • the coding region of the target gene was codon-optimized and had 5'/3' non-coding region sequences that could enhance the expression of the coding region.
  • Direct in vitro synthesis of mRNA has the characteristics of poor stability and strong immunogenicity.
  • the present invention adopts the introduction of chemically modified bases 5'methylcytosine and pseudouracil in the process of in vitro transcription and synthesis of mRNA to improve the synthesized mRNA. mRNA stability, reducing immunogenicity and increasing its translation efficiency.
  • Cytokine expression was detected by enzyme-linked immunosorbent assay (ELISA) by in vitro cell transfection
  • the expression of IL10 was verified by transfecting the IL10 cmRNA synthesized in vitro into HEK 293T/MB49/RAW 264.7 cells, and detecting the expression of cell culture supernatant and intracellular cytokine IL10 by ELISA.
  • the constructed IL10 cmRNA of the present invention can express IL10 in cells, and the expression efficiency is high.
  • the secretion of IL10 is realized by adding a signal peptide. Although the signal peptide is also chemically modified, it does not affect the exocytosis of IL10.
  • a mouse subcutaneous tumor model was constructed.
  • macrophage-deficient mice were constructed to exclude the interference of factors that cause macrophages to secrete IL10.
  • Organ tissue detection of IL10 and other key cytokine levels The animal experiment results of the present invention show that only one injection of cytokine interleukin 10 chemically modifies messenger ribonucleic acid after modeling can achieve a better tumor suppressing effect. While not wishing to be bound by theory, this may be due to the better stability and lower immunogenicity of cytokine interleukin-10 chemically modified messenger ribonucleic acid relative to cytokine interleukin-10.
  • cytokine interleukin-10 Direct injection of the cytokine interleukin-10 can lead to its rapid metabolism in vivo or induce tumor-promoting effects.
  • chemically modified cytokine interleukin-10 chemically modified messenger RNA can stably express interleukin-10 in vivo and release sufficient concentration of interleukin-10 to further activate the downstream pathway of interleukin-10, thereby achieving tumor-inhibiting effect.
  • Example 1 A mouse bladder cancer subcutaneous tumor model to explore the dynamic changes of IL10 concentration during tumor growth
  • the tumor-bearing mouse subcutaneous bladder cancer model was used. The day of injection of tumor cells was used as the time starting point. Tumor tissues were collected at 5d, 7d, 9d, 12d, 14d, and 16d, respectively, and the IL10 protein concentration was determined by ELISA.
  • Example 2 Mouse bladder cancer subcutaneous tumor model to explore the effect of IL10 level on tumor growth in vivo
  • a tumor-bearing mouse subcutaneous bladder cancer model was used. After successful modeling, 50 ⁇ g of IL10 antibody was injected through the tail vein of mice to neutralize IL10 in mice at -3d, 0d, 2d, 6d, 9d, 12d, and 14d, respectively. Mice injected intravenously with Phosphate Buffer Saline (PBS) were used as a control group to observe tumor growth.
  • PBS Phosphate Buffer Saline
  • Example 3 RAW 264.7 cells and Jurkat cells were cultured in vitro and added with different concentrations of IL10 to measure the mRNA transcript levels of IL10 receptors
  • IL10 can stimulate the transcription of the corresponding IL10 receptor gene and the expression of the corresponding protein in the macrophage cell line and T lymphocyte line at a certain concentration
  • the cultured RAW 264.7 cells and Jurkat cells were added containing different concentrations of IL10 (0 , 0.1, 0.5, 1, 3, 5, 7, 9, 10ng/mL) fresh medium, collected cells to extract RNA after culturing for 24h, and determined the content of IL10 receptor mRNA stimulated and activated by IL10.
  • IL10 cmRNA cytokine interleukin 10
  • RNA was synthesized by in vitro transcription reaction.
  • the m7G cap structure at the 5' end and the polyadenylic acid (polyA) structure at the 3' end were introduced to make it have a specific structure of mRNA, and 5' was introduced at the same time.
  • Chemically modified bases such as 'methylcytosine and pseudouracil' improve their stability and translation efficiency.
  • the in vitro transcription reaction system is 1 ⁇ g of linearized template DNA, 150nmol of adenine, 5'methylcytosine, pseudouracil, 120nmol of Anti-Reverse Cap Analog (ARCA) and 30nmol of guanine ribonucleoside acid.
  • T7 RNA polymerase was reacted at 37 degrees Celsius for 6 hours, DNase was added to continue the reaction at 37 degrees Celsius for 30 minutes to digest the DNA template, and then the reaction system was expanded to 100 ⁇ L, 100 nmol adenine and 250 nmol manganese chloride were added, and the temperature was 37 degrees Celsius.
  • Poly(A) polymerase was used to synthesize polyadenylic acid at the 3' end of RNA under the following conditions, and the tailing reaction was carried out for 1 hour.
  • Example 5 Determination of IL10 expression in MB49 cells and RAW 264.7 cells transfected with IL10 cmRNA
  • IL10 cmRNA In order to verify that IL10 cmRNA still has normal translation function in cancer cell lines and macrophage cell lines, 1 ⁇ g of IL10 cmRNA was transfected into bladder cancer cells MB49 and macrophage RAW 264.7 cells, respectively, and the cells were incubated for 12 h. At the starting point, the tissue fluid and cell supernatant were extracted at 12h, 24h, 48h, 60h, and 72h, respectively, and the expression of IL10 protein was determined by ELISA.
  • Example 2 From the results of Example 2 and Example 3, it can be seen that the present invention greatly prolongs the action time of cytokine IL10 by preparing chemically modified messenger RNA.
  • the action time of IL10 in vivo is very short, and the half-life is only a few hours, and from the results of the present invention, a substantially stable exocrine concentration was maintained at 60-80 hours.
  • the present invention adds a signal peptide for controlling the secretion sequence, the function of the signal peptide is not affected by chemical modification, the function of extracellular secretion is maintained, and high efficiency is achieved secretion.
  • Example 6 The mouse bladder cancer subcutaneous tumor model verifies the therapeutic effect of IL10 cmRNA tumor
  • a mouse bladder cancer subcutaneous tumor model was constructed for evaluation.
  • the tumor-bearing mouse subcutaneous bladder cancer model was used. After successful modeling, 10 ⁇ g or 40 ⁇ g IL10 cmRNA was injected into the tumor, and no IL10 cmRNA was injected as a negative control. Taking the day of injection as the time starting point, the body weight and tumor size of the mice were measured at 0d, 2d, 4d, 6d, 8d, 10d, and 14d. The results are shown in Figures 8-9.
  • Figure 8 shows the tumor growth in mice
  • the abscissa is the experimental time
  • the ordinate is the percentage of tumor volume increase, in which the volume of the tumor in the control group increased significantly over time, while the experimental group injected with 10 ⁇ g or 40 ⁇ g IL10 cmRNA Although the tumor The volume increased slightly, but the increase was lower, effectively inhibiting tumor development.
  • Figure 9 shows the changes in the body weight of the mice. The body weight of the mice in the experimental group injected with 10 ⁇ g or 40 ⁇ g of IL10 cmRNA was relatively stable after injection, which proved that the safety of IL10 cmRNA was high and the toxic and side effects were low.
  • the cycle of the animal experiment is longer than that of the above-mentioned cell experiment.
  • the present invention only injected IL10 cmRNA into the tumor-bearing mice once, it can be seen that it can act in vivo for a long time. It is also confirmed from the results of animal experiments that the IL10 cmRNA constructed by the present invention has a long half-life.
  • the experiments of the present invention have verified that the IL10 cmRNA of the present invention has an anti-tumor effect, possibly because the IL10 cmRNA of the present invention provides stably expressed high-concentration IL10, and a certain concentration of IL10 achieves downstream anti-tumor effects. Activation of tumor pathways.
  • the present invention overcomes the technical prejudice and confirms that the IL10 cmRNA of the present invention has an anti-tumor effect.

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Abstract

La présente divulgation concerne un réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine 10 et son application antitumorale, et concerne spécifiquement l'utilisation du réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine 10 dans la préparation de médicaments de prévention ou d'inhibition de tumeurs. Le réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine (10) est sélectionné parmi une quantité thérapeutiquement efficace d'interleukine 10, un médicament pouvant exprimer l'interleukine 10 chez un sujet, et un médicament pouvant stimuler la production d'interleukine 10 chez le sujet. L'invention concerne également un acide ribonucléique messager chimiquement modifié d'interleukine 10. L'acide ribonucléique messager est pourvu d'un cadre de lecture ouvert codant au moins pour une protéine d'interleukine 10, et l'acide ribonucléique messager est soumis à au moins une modification chimique. La présente invention surmonte le préjudice technique et trouve que l'interleukine 10 peut être utilisée pour inhiber les tumeurs avec des récepteurs de l'interleukine 10, et l'acide ribonucléique messager chimiquement modifié d'interleukine 10 présente un potentiel d'inhibition tumorale.
PCT/CN2020/142167 2020-12-31 2020-12-31 Réactif pouvant fournir une quantité thérapeutiquement efficace d'interleukine 10 et son application antitumorale WO2022141406A1 (fr)

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CN109628442A (zh) * 2019-01-08 2019-04-16 深圳市臻质医疗科技有限公司 mRNA及其制备方法和应用

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