WO2022141406A1

WO2022141406A1 - Reagent capable of providing therapeutically effective amount of interleukin 10 and anti-tumor application thereof

Info

Publication number: WO2022141406A1
Application number: PCT/CN2020/142167
Authority: WO
Inventors: 刘陈立; 陈茜; 董宇轩; 郭旋; 胡勇; 张昊
Original assignee: 深圳先进技术研究院
Priority date: 2020-12-31
Filing date: 2020-12-31
Publication date: 2022-07-07

Abstract

Disclosed are a reagent capable of providing a therapeutically effective amount of interleukin 10 and an anti-tumor application thereof, and specifically disclosed is use of the reagent capable of providing a therapeutically effective amount of interleukin 10 in preparation of drugs for preventing or inhibiting tumors. The reagent capable of providing a therapeutically effective amount of interleukin 10 is selected from a therapeutically effective amount of interleukin 10, a drug capable of expressing interleukin 10 in a subject, and a drug capable of stimulating production of interleukin 10 in the subject. Also provided is an interleukin 10 chemically modified messenger ribonucleic acid. The messenger ribonucleic acid is provided with an open reading frame encoding at least one interleukin 10 protein, and the messenger ribonucleic acid is subjected to at least one chemical modification. The present invention overcomes the technical prejudice and finds that the interleukin 10 can be used for inhibiting tumors with interleukin 10 receptors, and the provided interleukin 10 chemically modified messenger ribonucleic acid has tumor inhibition potential.

Description

A kind of reagent capable of providing therapeutically effective amount of interleukin 10 and its anti-tumor application

technical field

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.

Background technique

As a malignant disease, the number of patients with tumor is increasing year by year. The current conventional tumor treatment methods (surgery, radiotherapy, chemotherapy, etc.) all damage the normal tissues of the body and are accompanied by serious toxic and side effects, which cannot meet the growing clinical needs of patients.

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.

Transcription is a process that occurs in organisms all the time, and it is the first step from genetic information DNA to protein synthesis. In a cell-free system in vitro, 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. Synthesizing messenger ribonucleic acid (mRNA) in vitro and infusing it back into the body, and expressing the corresponding functional protein in vivo can be used for disease treatment, but due to the instability of mRNA itself (easy to be degraded by RNase) and immunogen Sex limits its application.

SUMMARY OF THE INVENTION

In order to solve the above problems, 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.

In the technical scheme of the present invention, 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;

In the technical solution of the present invention, 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;

In the technical solution of the present invention, 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, γδT cells, Drugs that synthesize and secrete interleukin-10 by any one or more of NK cells, mast cells, neutrophils, and eosinophils. For example, lipopolysaccharide, catecholamines, CD36 and p38 mitogen-activated protein (MAP) kinases.

In the technical solution of the present invention, 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.

In the technical solution of the present invention, 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.

In the technical solution of the present invention, 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; non-solid tumors selected from leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, mutated chronic myeloid leukemia, acute Lymphocytic leukemia.

In the technical solution of the present invention, 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, γδ 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.

In the technical scheme of the present invention, 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.

In the technical scheme of the present invention, 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.

In the technical scheme of the present invention, 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.

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.

In the technical solution of the present invention, 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, γδ T cells, NK cells, mast cells, neutrophils and eosinophils.

In the technical scheme of the present invention, 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 leukemia, acute myeloid leukemia, mutated chronic myeloid leukemia, acute lymphocytic leukemia.

In the technical solution of the present invention, 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;

In the technical scheme of the present invention, the method of administration includes intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration;

beneficial effect

1) The present invention finds that interleukin-10 can be used to inhibit tumors with interleukin-10 receptors. In the prior art, it is generally believed that 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.

2) 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. Activation of interleukin-10 receptors to induce downstream pathways.

3) 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.

4) The present invention greatly prolongs the half-life of interleukin-10 by constructing a messenger RNA chemically modified by interleukin-10. Compared with deoxyribonucleic acid, the messenger ribonucleic acid of the present invention does not need a transcription link and has a quick effect. Moreover, compared with the direct injection of interleukin 10 or the use of deoxyribonucleic acid for translation, the chemically modified messenger RNA of the present invention has less immunoreactivity and is more suitable for human or animal administration.

Description of drawings

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.

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.

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.

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.

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 μg 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 μg/40 μg 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 μg/40 μg 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.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below, but should not be construed as limiting the scope of the present invention.

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).

The terms "tumor inhibiting", "anti-tumor", "tumor inhibiting" and the like refer 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.

In some embodiments of the present invention, 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.

In some preferred embodiments of the present invention, 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.

In some preferred embodiments of the present invention, 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.

In some embodiments of the present invention, 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. Preferably, 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.

In some embodiments of the present invention, 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, γδ T cells, NK cells, mast cells, neutrophils and eosinophils.

In some embodiments of the invention, the above methods of administration include intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). Preferably, an agent capable of providing a therapeutically effective amount of interleukin 10 is applied directly within or near the tumor. Preferably, an agent capable of providing a therapeutically effective amount of interleukin 10 is targeted for delivery to the tumor site by a targeted formulation.

In some preferred embodiments of the present invention, 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.

In some embodiments of the invention, 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.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, 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, γδ 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.

In some preferred embodiments of the present invention, 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.

In some embodiments of 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 the sole active ingredient.

In some embodiments of the present invention, 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, glioblastoma; leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, mutated chronic myeloid leukemia, acute lymphocytic leukemia leukemia.

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.

In some embodiments of the invention, the open reading frame is codon-optimized.

In some embodiments of the present invention, the interleukin-10 is human-derived interleukin-10 and mouse-derived interleukin-10.

In some embodiments of the present invention, the interleukin 10 has homology with sequences NC_000001.11, NM_010548.270%-100%, preferably 95%-100%, or 99%-100% homology sex.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, the chemical modification of uracil is pseudouracil.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, the chemical modification of the cytosine is 5' methylcytosine.

In some embodiments of the present invention, 100% of the cytosines in the open reading frame are chemically modified to 5' methylcytosine; 100% of the uracils are chemically modified to pseudouracil.

In some embodiments of the invention, the open reading frame has upstream and or downstream non-coding sequences that enhance the expression of the open reading frame.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, the open reading frame further comprises a sequence encoding a signal peptide.

Other embodiments of the present invention provide a composition comprising the above chemically modified messenger ribonucleic acid.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, the cationic polymer is selected from the group consisting of Dendrimers and Polyethylenimine (PEI).

Another aspect of the present invention provides 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.

In some embodiments of the invention, 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.

In some embodiments of the present invention, 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.

In a preferred embodiment of the present invention, the drug capable of expressing interleukin 10 in a subject is selected from the above chemically modified messenger RNA or the above vector.

In some embodiments of the present invention, the above-mentioned antitumor pharmaceutical composition further includes any one of pharmaceutical excipients.

In some embodiments of the present invention, 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.

In some embodiments of the present invention, linear DNA is used as a template during the in vitro transcription reaction, and the linear DNA is DNA encoding any IL10.

In some embodiments of the present invention, adenine ribonucleotides, 5' methylcytosine adenine ribonucleotides, pseudouracil adenine ribonucleotides, and anti-reverse caps are added during the in vitro transcription reaction. and guanine ribonucleotides.

In a specific embodiment of the present invention, utilize chemically modified base to synthesize cytokine interleukin 10 messenger ribonucleic acid (IL10 cmRNA) in vitro, and the concrete method is as follows:

First, 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. In order to solve this problem, 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. At the same time, 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.

Testing the tumor therapeutic effect of the cytokine IL10 cmRNA in a tumor-bearing mouse model

In order to prove the inhibitory effect of IL10 cmRNA on the growth of solid tumors, a mouse subcutaneous tumor model was constructed. At the same time, 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. Direct injection of the cytokine interleukin-10 can lead to its rapid metabolism in vivo or induce tumor-promoting effects. However, 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.

The results are shown in Figure 1 and Figure 2. As the tumor continues to grow, the intratumoral IL10 concentration will first increase and then decrease. It is speculated that maintaining a high intratumoral IL10 concentration has a positive effect on tumor treatment. In the prior art, a fixed time point is usually used to analyze the expression level of IL10 in tumor tissue or a subject, so as to judge the tumor development or its tumor-promoting effect. However, through the experiments of the present invention, it is found that the level of IL10 in the tumor at different time points does not rise or fall all the time, but first rises and then falls. Comparing the results of tumor volume and IL10, it was found that the tumor volume grew rapidly with the decrease of IL10 concentration. It may be because the initial endogenous secreted IL10 has a certain effect of inhibiting tumors, but with the development of tumors, the concentration of IL10 is not enough to achieve tumor inhibition.

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 μg 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.

The results are shown in Figure 3. Compared with the control group, the tumor growth of mice injected with IL10 antibody was not significantly affected and continued to grow. The experimental results first confirmed that IL10 is not a tumor-promoting factor, and reducing the level of IL10 in vivo did not reduce the occurrence and development of tumors. On the contrary, the tumor volume was larger than that of the control group at 12 and 14 days. The experimental results are combined with Example 5 The results showed that the endogenous secretion of IL10 inside the tumor decreased over time and the combined effect of IL10 antibody resulted in a larger tumor volume in the later stage compared with the control group.

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

In order to verify that 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.

The results are shown in Figure 4 and Figure 5. Adding low concentrations of IL10 could not stimulate the transcription of the IL10 receptor gene, but when the added IL10 reached and exceeded a certain concentration (RAW 264.7>1ng/mL, Jurkat>3ng/mL), IL10 The receptor mRNA level increased rapidly, indicating that the binding of IL10 to the IL10 receptor stimulates the signal transduction of the downstream pathway, and the transcription of the IL10 receptor gene is activated. The experimental results are also consistent with the results of Examples 5-6.

Example 4 Construction of cytokine interleukin 10 (IL10) chemically modified messenger ribonucleic acid (IL10 cmRNA)

Using the DNA of IL10 as a template, the DNA of IL10 was constructed on a plasmid with a T7 promoter, and the upstream and downstream of the IL10 coding region had sequences such as 5'/3' non-coding regions to enhance expression. After linearizing it as a template, RNA was synthesized by in vitro transcription reaction. In the synthesis 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 1μg of linearized template DNA, 150nmol of adenine, 5'methylcytosine, pseudouracil, 120nmol of Anti-Reverse Cap Analog (ARCA) and 30nmol of guanine ribonucleoside acid. After 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 μL, 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. Add 150 μL of nuclease-free ultrapure water and 150 μl of lithium chloride precipitant to each 100 μL reaction system to purify the synthesized mRNA, react at -20 degrees Celsius for at least 2 hours, centrifuge, wash with 70% ethanol and dissolve in nuclease-free ultrapure water , store in -80 degrees Celsius refrigerator.

The results are shown in Figure 6. The target length of IL10 cmRNA was successfully synthesized by in vitro transcription, and the tailing was successful at the same time (lane 1 is the untailed RNA, lane 2 is the tailed RNA, and the reference is RiboRuler High Range RNA Ladder).

Example 5 Determination of IL10 expression in MB49 cells and RAW 264.7 cells transfected with IL10 cmRNA

In order to verify that IL10 cmRNA still has normal translation function in cancer cell lines and macrophage cell lines, 1 μg 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.

The results are shown in Figure 7. Compared with the control group of cells without IL10 cmRNA transfection, the expression of IL10 in the supernatants of the two cell cultures transfected with IL10 cmRNA was at a relatively high level, while the expression of IL10 in the tissue fluid of the cells was at a relatively high level. The expression of IL10 was low, indicating that IL10 was normally translated and secreted into the extracellular space.

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. In the prior art, 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.

In addition, it can be seen from the results of Examples 2 and 3 that 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

In order to verify the anti-tumor effect of IL10 cmRNA, 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 μg or 40 μg 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, and 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 μg or 40 μg 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 μg or 40 μg 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. Although 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. Although not wishing to be bound by theory, 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.

Claims

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;

Preferably, 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;

More preferably, the therapeutically effective amount of interleukin 10 is selected from long-circulating, actively targeting tumor and or low-degraded interleukin 10; The preparation is obtained by loading interleukin-10, and or interleukin-10 obtained by active targeting and or long-circulating chemical modification; the low-degraded interleukin-10 is an inhibitor that inhibits interleukin-10, or the inhibitor is used in combination with interleukin-10;

More preferably, 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 carrier is selected from an aqueous solution, a saline solution , buffer, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector, microcyclic vector or chemically synthesized vector;

More preferably, 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, γδ T cells, NK cells, mast cells, A drug that synthesizes and secretes interleukin-10 by any one or more of neutrophils and eosinophils.
Use of an agent capable of providing a therapeutically effective amount of interleukin-10 in the preparation of a medicament for activating an interleukin-10 receptor or a pathway downstream of an interleukin-10 receptor to inhibit tumors;

Preferably, 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;

More preferably, the therapeutically effective amount of interleukin 10 is selected from long-circulating, actively targeting tumor and or low-degraded interleukin-10; The preparation is obtained by loading interleukin-10, and or interleukin-10 obtained by active targeting and or long-circulating chemical modification; the low-degraded interleukin-10 is an inhibitor that inhibits interleukin-10, or the inhibitor is used in combination with interleukin-10;

More preferably, 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 carrier is selected from an aqueous solution, a saline solution , buffer, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector, microcyclic vector or chemically synthesized vector;

More preferably, 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, γδ T cells, NK cells, mast cells, A drug that synthesizes and secretes interleukin-10 by any one or more of neutrophils and eosinophils.
Use of an agent capable of providing a therapeutically effective amount of interleukin 10 in preventing or treating tumors;

Preferably, 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;

More preferably, the therapeutically effective amount of interleukin 10 is selected from long-circulating, actively targeting tumor and or low-degraded interleukin 10; The preparation is obtained by loading interleukin-10, and or interleukin-10 obtained by active targeting and or long-circulating chemical modification; the low-degraded interleukin-10 is an inhibitor that inhibits interleukin-10, or the inhibitor is used in combination with interleukin-10;

More preferably, 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 carrier is selected from an aqueous solution, a saline solution , buffer, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector, microcyclic vector or chemically synthesized vector;

More preferably, 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, γδ T cells, NK cells, hypertrophy A drug that synthesizes and secretes interleukin 10 by any one or more of cells, neutrophils, and eosinophils.
The use according to any one of claims 1-3, wherein 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 the subject. reagent;

Preferably, 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 secrete interleukin endogenously, and the concentration of the secreted interleukin reaches the level of the drug that activates the transcription level of the interleukin 10 receptor.
The use according to any one of claims 1-3, wherein the tumor is a tumor expressing interleukin 10 receptor on the surface of immune cells,

Preferably bladder tumor, colorectal tumor, breast tumor, melanoma, testicular tumor, prostate tumor, nephroma, 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, esophageal 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; leukemia , chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, mutated chronic myeloid leukemia, acute lymphoblastic leukemia.
A chemically modified messenger RNA, characterized in that 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;

Preferably, the chemical modification is selected from the base modification of nucleotides as 5'methylcytosine, pseudouracil, N6-methyladenosine, inosine, 5 hydroxymethylcytosine, N1methyladenosine glycoside, 2-thiouracil, 2-thiocytosine, 3-methylcytosine, 5-hydroxycytosine, 5-bromocytosine, 5-fluorocytosine, 5-chlorocytosine, 6-azo Cytosine, azacytosine, 5,6-dihydrocytosine, N4-ethylcytosine, 5-bromouracil, 5-chlorouracil, 5-fluorouracil, 5-iodouracil, 5-methyl At least one of uracil, 5-methyl-2-thiouracil, 2-thiouracil, and 4-thiouracil.
The chemically modified messenger ribonucleic acid according to claim 5, wherein the open reading frame further comprises a sequence encoding a signal peptide.
A composition, characterized in that the composition comprises the chemically modified messenger ribonucleic acid according to any one of claims 6-7;

Preferably, 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 vector, mRNA vector, microcircle or chemically synthesized vector.
An anti-tumor pharmaceutical composition, characterized in that the anti-tumor pharmaceutical composition comprises a therapeutically effective amount of the chemically modified messenger ribonucleic acid according to any one of claims 6-7 or the combination according to claim 8 thing;

Preferably, 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, brain tumor, ovarian tumor, small tumor 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 sinus cancer, nasopharyngeal cancer cancer, oral 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; leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, acute myeloid leukemia, mutated chronic myeloid leukemia, acute lymphocytic leukemia.
The use according to any one of claims 1-5, wherein the agent capable of providing a therapeutically effective amount of interleukin 10 is selected from the chemically modified messenger ribonucleic acid according to any one of claims 6-7, The composition of claim 8 or the antitumor pharmaceutical composition of claim 9.
A method for treating or preventing tumors, characterized in that the method comprises the use of an agent capable of providing a therapeutically effective amount of interleukin 10 to a subject;

Preferably, the method of administration comprises intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration;

Preferably, 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;

More preferably, the therapeutically effective amount of interleukin 10 is selected from long-circulating, actively targeting tumor and or low-degraded interleukin 10; The preparation is obtained by loading interleukin-10, and or interleukin-10 obtained by active targeting and or long-circulating chemical modification; the low-degraded interleukin-10 is an inhibitor that inhibits interleukin-10, or the inhibitor is used in combination with interleukin-10;

More preferably, 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 carrier is selected from an aqueous solution, a saline solution , buffer, glucose solution, liposome, cationic liposome, cationic polymer, lysophospholipid, viral vector, plasmid vector, mRNA vector, microcyclic vector or chemically synthesized vector;

More preferably, 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, γδ T cells, NK cells, hypertrophy A drug that synthesizes and secretes interleukin 10 by any one or more of cells, neutrophils, and eosinophils.