WO2022110010A1 - Utilisation d'un activateur immunitaire, d'un antigène et d'un anticorps dans la préparation d'un produit pour la prévention et le traitement de tumeurs - Google Patents

Utilisation d'un activateur immunitaire, d'un antigène et d'un anticorps dans la préparation d'un produit pour la prévention et le traitement de tumeurs Download PDF

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WO2022110010A1
WO2022110010A1 PCT/CN2020/132222 CN2020132222W WO2022110010A1 WO 2022110010 A1 WO2022110010 A1 WO 2022110010A1 CN 2020132222 W CN2020132222 W CN 2020132222W WO 2022110010 A1 WO2022110010 A1 WO 2022110010A1
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time
oligonucleotide
administration
tumor
cpg
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PCT/CN2020/132222
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Chinese (zh)
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胡荣宽
李琴
张佩琢
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SUZHOU GENEPHARMA CO Ltd
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SUZHOU GENEPHARMA CO Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/711—Natural deoxyribonucleic acids, i.e. containing only 2'-deoxyriboses attached to adenine, guanine, cytosine or thymine and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity

Definitions

  • the invention relates to the field of cancer research, in particular to the application of an immune activator, an antigen and an antibody in the preparation of products for preventing and treating tumors.
  • Malignant melanoma is a common skin tumor caused by excessive proliferation of abnormal melanocytes. It is extremely malignant and accounts for a large proportion of skin tumor deaths. It occurs mostly in the skin or mucous membranes close to the skin, but also in the pia mater and choroid. It is a common malignant tumor of skin, mucous membranes and pigmented membranes, and one of the fastest growing malignant tumors, with an annual growth rate of 3%-5%. For a long time, surgery, radiotherapy and chemotherapy are the main methods for the treatment of melanoma, but these methods also have many shortcomings, such as trauma, large side effects and low patient tolerance.
  • peptide vaccines are easy to prepare, easy to store, and safe to use. They have become a hot spot for immunotherapy to treat tumors. They have been widely used in recent years, and can construct corresponding synthetic antigenic peptides against complex non-contiguous natural antigenic determinants.
  • the technical problem solved by the present invention is how to treat tumors.
  • the present invention provides any of the following applications:
  • the immune activator is CpG oligonucleotide; the antigen is M30 mRNA; the antibody is a monoclonal antibody, and the monoclonal antibody is PD1;
  • the M30 mRNA is obtained by in vitro transcription of DNA whose sequence is as shown in SEQ ID NO.2;
  • the CpG oligonucleotides are as follows 1) or 2)
  • the chemical modification is that one or more than one phosphodiester bond in the oligonucleotide is replaced by a phosphorothioate bond.
  • the tumor is melanoma, non-small cell lung cancer, breast cancer or colorectal cancer
  • the tumor cells are melanoma cells, non-small cell lung cancer cells, breast cancer cells or colorectal cancer cells.
  • the melanoma cells are B16 melanoma cells.
  • the administration mode of CpG nucleic acid is subcutaneous injection or intratumoral injection; the administration time of CpG nucleic acid is once every 2-4 days; preferably once every 3 days or twice a week; the dosage of CpG nucleic acid is: 1mg/kg/time-2.5mg/kg/time; preferably 2mg/kg/time-2.5mg/kg/time.
  • the administration mode of PD1 is intravenous injection; the administration time of PD1 is once every 2-4 days; preferably once every 3 days; the dosage of PD1 is 6mg/kg/time-10mg/kg/time; preferably 8mg /kg/time
  • the administration mode of M30 is intravenous injection; the administration time of M30 is 2-4 times/week; preferably, the administration time of M30 is 2 times/week; the administration dose of M30 is 0.5mg/kg/time-1.5mg /kg/time; preferably 0.8 mg/kg/time.
  • a pharmaceutical composition for the prevention, treatment or adjuvant treatment of tumors comprising an immune activator and an antigen
  • a pharmaceutical composition for the prevention, treatment or adjuvant treatment of tumors comprising an immune activator and an antibody;
  • the immune activator is CpG oligonucleotide; the antigen is M30 mRNA; the antibody is a monoclonal antibody, and the monoclonal antibody is PD1; the M30 mRNA is represented by the sequence as shown in SEQ ID NO.2 obtained by in vitro transcription of DNA;
  • the CpG oligonucleotides are as follows 1) or 2)
  • CpG2018B, PD1 and the combination of CpG2018B and PD1 can inhibit the increase of tumor volume; the combination of CpG2018B and PD1 has the best effect.
  • M30 mRNA, CPG2018B and the combination of M30 mRNA and CPG2018B can inhibit the increase of tumor volume and tumor weight; the combination of M30 mRNA and CPG2018B has the best effect.
  • Fig. 1 is the volume growth curve of the animal tumor body in Example 1;
  • Figure 2 is the tumor pictures of each group in Example 1; the control group in the figure refers to the NC group, 2018B refers to CpG-2018 administered alone; 2018B+PD1 refers to the combined administration of CpG-2018 and PD1;
  • Figure 3 is a picture of the animal tumor in Example 2; CpG refers to CpG-2018 administered alone; mRNA refers to M30 mRNA administered alone; combo refers to the combined administration of CpG-2018 and M30 mRNA;
  • Figure 4 is the tumor volume growth curve of the animal in Example 2; CpG refers to CpG-2018 administered alone; mRNA refers to M30 mRNA administered alone; combo refers to the combined administration of CpG-2018 and M30 mRNA;
  • Figure 5 is a histogram of tumor weights in each group in Example 2; M30 mRNA+CpG refers to the combined administration of M30 mRNA and CpG-2018.
  • CpG2018B was synthesized by Suzhou Gema Gene Co., Ltd. It is a short-chain nucleotide sequence, and its specific sequence is: TCGTCGTTTGACGTTTCGTTTG, and the phosphodiester bonds between all bases are phosphorothioate phosphodiester bonds.
  • PD-1 monoclonal antibody Daboshu sintilimab injection
  • Innovent Bio hereinafter referred to as PD1.
  • Example 1 CpG nucleic acid and/or PD1 inhibit tumor cell growth
  • marker scissors ophthalmic scissors, ophthalmic surgical forceps, vernier calipers and cameras;
  • Each rat cage wears an identification card with information such as the experiment number, experimental group, experimenter's name, mouse breed and gender, and the mice are marked with ear piercing.
  • the ambient temperature of the animal room was 22 ⁇ 3°C, the relative humidity was 40-70%, and the 12-hour light/12-hour dark cycle was performed.
  • the bedding material was autoclaved corncob bedding (Jiangsu Synergy Medicine Bioengineering Co., Ltd.), and the cages were changed once a week.
  • Feed was purchased from Jiangsu Synergy Bio.
  • the water used for experimental animals was sterilized by secondary reverse osmosis filtration.
  • Laboratory animals must be healthy and acclimatized, with no restriction on food and water.
  • B16 cells were cultured in a petri dish to 90% confluence, they were routinely digested with trypsin and washed twice with PBS to prepare a single cell suspension.
  • the single-cell resuspension was injected subcutaneously into the scapula of the experimental animals with a disposable syringe, and each mouse was subcutaneously inoculated with 0.1 mL of the single-cell resuspension.
  • IV intravenous injection
  • SC subcutaneous injection
  • PBS in group G1 represents PBS buffer
  • 20 ⁇ L in group G2 is CpG2018B dissolved in PBS buffer to obtain 20 ⁇ L drug solution
  • 100 ⁇ L in group G3 is PD-1 Diluted with PBS buffer to obtain 100 ⁇ L of drug solution
  • 20 ⁇ L of CpG2018B in the G4 group was dissolved in PBS buffer to obtain 20 ⁇ L of drug solution
  • 100 ⁇ L of PD-1 was diluted with PBS buffer to obtain 100 ⁇ L of drug solution.
  • SC+IV was: CpG2018B was administered in SC, and PD1 was administered by IV.
  • the experimental animals were sacrificed and the tumors were taken out.
  • the tumors were arranged according to the numbers, and the long and short diameters of the tumor were measured with a vernier caliper, weighed, recorded and photographed with scale pictures of each group of tumor tissues.
  • the tumor was divided into two parts, one part was fixed in formalin solution, and the other part was frozen at -80°C.
  • Day 11 Select a suitable tumor and start the formal experiment (called day 0). As shown in Table 1, the mice were divided into 4 groups, 5 mice/group, with a total of 20 female mice.
  • NC group was used as the control group. All experimental groups were administered once every three days and continued to be administered for 3 times. All of them had a significant inhibitory effect on the mouse B16 tumor model. Among them, the 2018B+PD1 group had the most effect. it is good.
  • Example 2 CpG nucleic acid and/or M30 inhibit tumor cell growth
  • B16 cells were routinely cultured in DMEM medium containing 10% FBS (Genial) at 37°C in a 5% CO 2 saturated humidity incubator.
  • B16 cells were cultured in a petri dish to 90% confluence, they were routinely digested with trypsin and washed twice with PBS to prepare a single cell suspension.
  • the single-cell resuspended solution was injected subcutaneously into the scapula of experimental animals with a disposable syringe, and 0.1 mL of the single-cell resuspended solution was subcutaneously inoculated in each mouse.
  • Group A CpG-2018, 2.5mg/kg/time, day 0, 3, 5, 7, 9, IT;
  • Group B M30 mRNA, 0.8mg/kg/time, day 0, 3, 5, 7, 9, IV;
  • Group C CpG-2018 combined with M30 mRNA; M30 mRNA 0.8 mg/kg/time, CpG-2018 2.5 mg/kg/time; administration time is day 0, 3, 5, 7, 9; M30 mRNA: IV; CpG: IT
  • Group D Negative control (PBS), IT;
  • IV intravenous injection; IT intratumoral injection; the injection volume of CpG-2018 per mouse is the same as in Example 1, that is, CpG-2018 is dissolved in 20 ⁇ L PBS buffer for injection; the injection volume of M30 mRNA per mouse is 20 ⁇ L, that is, dissolve M30 mRNA in 20 ⁇ L PBS buffer for injection.
  • Day 11 Select a suitable tumor and start the formal experiment (called day 0); divide into 4 groups, 5 mice/group, with a total of 20 mice.
  • Day 22 (day 11): The tumor was killed and the tumor was taken out. The tumor was placed according to the number. The length and transverse diameter of the tumor were measured with a vernier caliper, weighed, recorded and photographed with a ruler for each group of tumor tissue.
  • the tumor was divided into two parts, one part was fixed in formalin solution, and the other part was frozen at -80°C.

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Abstract

L'invention concerne l'une quelconque des utilisations suivantes : 1) l'utilisation d'un activateur immunitaire et/ou d'un antigène dans la préparation d'un produit pour la prévention, le traitement ou le traitement auxiliaire de tumeurs ; 2) l'utilisation d'un activateur immunitaire et/ou d'un anticorps dans la préparation d'un produit pour la prévention, le traitement ou le traitement supplémentaire de tumeurs ; 3) l'utilisation d'un activateur immunitaire et/ou d'un antigène dans la préparation d'un produit pour inhiber la prolifération ou la croissance de cellules tumorales ; et 4) l'utilisation d'un activateur immunitaire et/ou d'un anticorps dans la préparation d'un produit pour inhiber la prolifération ou la croissance de cellules tumorales. L'activateur immunitaire est un oligonucléotide CpG ; l'antigène est un ARNm M30 ; l'anticorps est un anticorps monoclonal PD1 ; l'ARNm M30 est obtenu au moyen de la transcription in vitro de l'ADN avec une séquence telle que représentée dans SEQ ID No 2 ; et l'oligonucléotide CpG est le suivant : 1) un oligonucléotide tel que représenté dans SEQ ID No 1 ; ou 2 ) un oligonucléotide obtenu par modification chimique de l'oligonucléotide tel que représenté dans SEQ ID No 1.
PCT/CN2020/132222 2020-11-27 2020-11-27 Utilisation d'un activateur immunitaire, d'un antigène et d'un anticorps dans la préparation d'un produit pour la prévention et le traitement de tumeurs Ceased WO2022110010A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
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CN116712541A (zh) * 2023-07-26 2023-09-08 安徽大学 一种用于抗肿瘤的联合用药物组合物及其应用

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CN107430132A (zh) * 2015-02-12 2017-12-01 生物技术Rna制药有限公司 预测可用于疫苗接种的t细胞表位
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116712541A (zh) * 2023-07-26 2023-09-08 安徽大学 一种用于抗肿瘤的联合用药物组合物及其应用

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