WO2026005647A2 - Agent immunobiologique pour traiter des maladies oncologiques sur la base d'un vecteur arn - Google Patents

Agent immunobiologique pour traiter des maladies oncologiques sur la base d'un vecteur arn

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Publication number
WO2026005647A2
WO2026005647A2 PCT/RU2025/000257 RU2025000257W WO2026005647A2 WO 2026005647 A2 WO2026005647 A2 WO 2026005647A2 RU 2025000257 W RU2025000257 W RU 2025000257W WO 2026005647 A2 WO2026005647 A2 WO 2026005647A2
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seq
reading frame
mrna
open reading
sequence seq
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Russian (ru)
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WO2026005647A3 (fr
Inventor
Александр Леонидович ГИНЦБУРГ
Андрей Дмитриевич КАПРИН
Денис Юрьевич ЛОГУНОВ
Владимир Алексеевич ГУЩИН
Амир Ильдарович ТУХВАТУЛИН
Наталья Михайловна Тухватулина
Софья Рубеновна КОЗЛОВА
Наталия Петровна АНТОНОВА
Елена Владимировна ШИДЛОВСКАЯ
Бахтияр Исраил оглы ВЕРДИЕВ
Роман Алексеевич ИВАНОВ
Денис Александрович КЛЕЙМЕНОВ
Евгения Николаевна БЫКОНЯ
Елена Петровна МАЗУНИНА
Алина Шахмировна ДЖАРУЛЛАЕВА
Андрей Андреевич ПОЧТОВЫЙ
Андрей Эдуардович СИНЯВИН
Евгений Валерьевич УСАЧЕВ
Дарья Владимировна ВАСИНА
Надежда Анатольевна КУЗНЕЦОВА
Сергей Евгеньевич ДМИТРИЕВ
Игорь Андреевич ИВАНОВ
Ирина Леонидовна ТУТЫХИНА
Тимофей Андреевич РЕМИЗОВ
Анастасия Андреевна ЗАХАРОВА
Александр Сергеевич Семихин
Андрей Игоревич ГОСУДАРЕВ
Дмитрий Николаевич ЩЕРБИНИН
Елизавета Александровна ТОКАРСКАЯ
Надежда Леонидовна ЛУБЕНЕЦ
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Federal State Budgetary Institution "national Research Center Of Epidemiology And Microbiology Named After Honorary Academician NF Gamalei" Of Ministry Of Health Of Russian Federation
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Federal State Budgetary Institution "national Research Center Of Epidemiology And Microbiology Named After Honorary Academician NF Gamalei" Of Ministry Of Health Of Russian Federation
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Priority claimed from RU2024118081A external-priority patent/RU2828631C1/ru
Application filed by Federal State Budgetary Institution "national Research Center Of Epidemiology And Microbiology Named After Honorary Academician NF Gamalei" Of Ministry Of Health Of Russian Federation filed Critical Federal State Budgetary Institution "national Research Center Of Epidemiology And Microbiology Named After Honorary Academician NF Gamalei" Of Ministry Of Health Of Russian Federation
Publication of WO2026005647A2 publication Critical patent/WO2026005647A2/fr
Publication of WO2026005647A3 publication Critical patent/WO2026005647A3/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • An immunobiological agent for the treatment of oncological diseases based on an mRNA vector.
  • the invention relates to immunology and oncology.
  • the developed immunobiological agent can be used in the treatment of patients with cancer of various etiologies.
  • the proposed invention expands the arsenal of means and methods for inducing an immune response in cancer patients.
  • Cancer is one of the leading causes of death worldwide. According to the WHO, in 2022 there will be 20 million new cases of cancer and 9.7 million deaths from cancer worldwide. The number of people alive 5 years after diagnosis in 2022 was 53.5 million. According to these data, approximately one in five people will develop some kind of cancer during their lifetime; about 1 in 9 men and 1 in 12 women will die from this disease [“The Global Cancer Burden Is Growing Along with the Rising Need for Services.” World Health Organization, World Health Organization, www.who.int/ru/news/item/01-02-2024-global-cancer-burden-growing— amidst-mounting-need-for-services. Accessed: 18 June 2024].
  • Cancer development involves the progressive transformation of normal cells into malignant ones, which is the result of genetic and epigenetic changes occurring within the cells.
  • tumor cells can be eliminated by effector cells of the immune system.
  • immune selection tumor immunoediting
  • soluble factors secreted by tumor cells can create a tumor-like environment. the microenvironment provides favorable conditions for evading the effects of the immune system.
  • Tumor cells are known to suppress the targeted immune response using various defense mechanisms. Some of these are associated with the activation of a system of inhibitory mechanisms (immune checkpoints), which transmit an inhibitory signal to cytotoxic T lymphocytes, thereby suppressing immunological reactivity in the initial, inductive phase of the immune response. As a result, T lymphocyte activation is blocked, leading to the predominance of T regulatory cells in the tumor microenvironment and the development of immune tolerance and anergy [Shubnikova E.V., Bukatina T.M., Velts N.Yu., Kaperko D.A., Kutekhova G.V. Immune checkpoint inhibitors: new risks of a new class of antitumor agents // Safety and Risk of Pharmacotherapy, 2020, No. 1, pp. 9-20].
  • inhibitory mechanisms immunological reactivity in the initial, inductive phase of the immune response.
  • immune checkpoint inhibitors have revolutionized oncoimmunology.
  • a whole class of drugs consisting of humanized monoclonal antibodies that inhibit immune checkpoints, has now been developed.
  • This class of drugs has significantly improved patient survival and has become a major breakthrough in anticancer therapy.
  • four immune checkpoint inhibitors are registered in Russia: ipilimumab, nivolumab, pembrolizumab, and atezolizumab.
  • this field is actively developing, and new targets are being sought.
  • this class of drugs cannot address a number of issues related to low immunogenicity and insufficient presentation of tumor antigens. As a result, some tumor cells are able to evade the immune response.
  • Dendritic cell (DC)-based vaccines are dendritic cells (DC) act as antigen-presenting cells in the human immune system. In this type of vaccine, DC enhance the presentation of tumor antigens to lymphocytes. After activating naive lymphocytes, they become capable of destroying tumor cells presenting these antigens [Bozhenko V.K. Antitumor vaccines. Bulletin of the Russian Scientific Center of Roentgenology and Radiology of the Ministry of Health of the Russian Federation 2022, No. 1, Vol. 22]. Anticancer vaccines of this type typically include DC isolated from patients or generated ex vivo by culturing the patient's hematopoietic progenitor cells or monocytes.
  • DCs are further loaded with tumor antigens and sometimes combined with immunostimulatory agents such as GM-CSF.
  • immunostimulatory agents such as GM-CSF.
  • Another class of cancer vaccines is based on modified (e.g., irradiated with sublethal doses) tumor cells used as antigens, also in combination with immunostimulatory agents.
  • Vaccines of this type are based on both autologous (e.g., OncoVAX, LipoNova) and allogeneic (e.g., Canvaxin, Onyvax-P, GV AX) tumor cell lines.
  • autologous e.g., OncoVAX, LipoNova
  • allogeneic e.g., Canvaxin, Onyvax-P, GV AX
  • the use of these vaccination methods allows for the production of a reproducible, safe vaccine product in which the injected tumor cells cannot proliferate. Irradiated tumor cells naturally express numerous specific antigens, thus facilitating the initiation of an antitumor immune response.
  • whole-cell vaccines can be modified to enhance immunogenicity by transfecting immunostimulatory molecules.
  • the GVAX vaccine platform involves the use of irradiated allogeneic tumor cell lines modified to secrete GM-CSF to enhance the immunogenicity of the vaccine.
  • irradiated cells when cells are irradiated, phosphatidyl serine, an immunosuppressive phospholipid normally found on the inner leaflet of the plasma membrane, is translocated from the inner side of the plasma membrane to its outer side, which in turn leads to the secretion of immunosuppressive factors by dendritic cells and inhibits their maturation, promoting an immunosuppressive environment for the tumor.
  • irradiated cells may retain the ability to secrete immunosuppressive factors similar to the original tumor cells.
  • radiation-induced suppression immunity partially negates the immunogenic effect of irradiated whole cells [Srivatsan S, Patel JM, Bozeman EN, Imasuen IE, He S, Daniels D, Selvaraj P.
  • the next class of therapeutic antitumor vaccines is based on peptide fragments of antigens selectively expressed by tumor cells.
  • the peptides are administered alone or in combination with immunostimulatory agents, which may include adjuvants and cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • immunostimulatory agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF).
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • Peptides loaded into MHC class I are recognized by specific TCRs on CD8+ T cells, which are activated to exert their cytotoxic activity against tumor cells presenting the same peptide-MHC-I complex. This process is defined as active immunotherapy, as the host immune system is either activated de novo or restimulated to trigger an effective tumor-specific immune response, which can ultimately lead to tumor regression.
  • the objective of the present invention is to expand the arsenal of means for the treatment of oncological diseases.
  • the technical result consists in the development of a means that allows for the effective induction of an immune response against tumor cells.
  • an immunobiological agent for the therapy of oncological diseases has been created based on an mRNA vector, in which the mRNA contains the sequence SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:8 at the 5'end; an open frame reads encoding from 1 to 80 tumor gene signatures; sequence SEQ ID N0:6 or SEQ ID N0:7 at the 3' end.
  • the technical result consists in the development of a method for producing an immunobiological agent, comprising the following stages: a) identifying gene signatures of tumor cells; b) synthesizing a set of DNA matrices, each of which contains an open reading frame encoding 1 to 80 detected gene signatures; c) synthesizing a set of mRNA based on the obtained set of DNA matrices;
  • the developed immunobiological agent can be used to induce an immune response to tumor cells that contain gene signatures included in the mRNA vector.
  • Fig. 1 shows the results of in vivo bioluminescence measurements using the IVIS Imaging System (Perkin Elmer, USA).
  • mRNA that has the sequence SEQ ID N0:1 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:2 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:3 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:4 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:5 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:8 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame 7. mRNA that has the sequence SEQ ID N0:1 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:2 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:3 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:4 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:5 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:8 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • Fig. 2 shows the results of measuring the tumor volume in animals that were administered a phosphate-buffered solution and the studied mRNA preparations.
  • the ordinate axis shows the tumor volume, mm 3 .
  • mice that were injected with phosphate-buffered saline
  • mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:12;
  • mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:17;
  • mice injected with mRNA containing an open reading frame encoding the 3 identified gene signatures of SEQ ID N0:20.
  • Fig. 3 shows the results of measuring the tumor volume in animals that were administered a phosphate-buffered solution and the studied mRNA preparations.
  • the ordinate axis shows the tumor volume, mm 3 .
  • the abscissa axis shows different groups of animals, where
  • Fig. 4 shows the results of determining the cellular composition of the tumor microenvironment
  • mice 1 - intact mice (no drug was administered), 2 - the drug under study.
  • tumor cells adapt by acquiring mutations and altering the levels of rare proteins essential, for example, for cell survival. This can lead to changes in the molecular signatures of antigens present on tumor cells, which the immune system can recognize. Tracking these changes can reveal new targets for therapeutic agents. However, each malignancy has its own unique characteristics. Therefore, the genetic signatures of tumor cells can vary significantly between patients, even with the same tumor location.
  • the developed method for producing an immunobiological agent involves sequencing DNA isolated from a patient's tumor cells, which results in the creation of a mutaname—a collection of somatic cancer mutations in a specific tumor.
  • a unique algorithm is used to identify gene signatures specific to a patient's tumor cells (i.e., specific changes in gene expression characteristic of a given patient's tumor cells).
  • the ultimate goal of identifying gene signatures is to identify highly immunogenic antigens strictly specific to tumor cells.
  • a set of DNA matrices containing an open reading frame encoding the identified gene signatures is then created.
  • a single DNA matrix contains an open reading frame encoding a single identified gene signature.
  • a single DNA matrix contains an open reading frame encoding between 2 and 80 identified gene signatures.
  • the maximum number of gene signatures in a single DNA template (80) is determined by the maximum capacity of the genetic construct. This number also depends on how many specific gene signatures were detected in a particular patient's tumor.
  • the DNA template contains all the building blocks for the transcription of functional mRNA, as well as the elements necessary for plasmid growth in bacterial cell culture.
  • mRNA that has the sequence SEQ ID N0:2 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame mRNA that has the sequence SEQ ID N0:3 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:4 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:8 before the open reading frame and has the sequence SEQ ID N0:6 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:1 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • mRNA that has the sequence SEQ ID N0:3 before the open reading frame and has the sequence SEQ ID N0:7 after the open reading frame.
  • Example 5 demonstrates that all of the listed mRNA constructs are capable of providing mRNA translation and target protein synthesis.
  • the above-mentioned mRNAs were created, containing various constructs at the 5' (SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:8) and 3' ends (SEQ ID N0:7, SEQ ID N0:7), with an open reading frame encoding luciferase.
  • SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:8) and 3' ends (SEQ ID N0:7, SEQ ID N0:7) with an open reading frame encoding luciferase.
  • mRNAs obtained by in vitro transcription using the developed DNA templates were subsequently packaged into lipid nanoparticles and administered into mammals.
  • Translation of mRNA containing an open reading frame encoding gene signatures specific to tumor cells has been shown to facilitate immune system recognition of tumor antigens.
  • the immune system effectively targets and destroys tumor cells. This effect has been shown to correlate with changes in the tumor microenvironment.
  • the invention is supported by the following examples.
  • Example 1 Identification of differences in the gene signatures of tumor and normal cells.
  • the search for genetic signatures of tumor and normal cells is based on obtaining genetic data characterizing the complete genome and/or exome (for normal and tumor cells) and transcriptome (for tumor cells). Nucleic acid extraction from samples and sequencing are performed using available commercial kits according to the manufacturer's instructions. The obtained data are analyzed using proprietary software that enables genomic data quality control, filtering and removal of low-quality sequencing data, mapping to a reference sequence, identifying germline and somatic mutations, searching for specific antigens, including bioinformatic prediction of peptide binding to major histocompatibility complex (MHC) molecules that can be presented on the cell surface and recognized by T cells, as well as assessing immunogenicity and a number of other parameters.
  • MHC major histocompatibility complex
  • the number of selected gene signatures can vary from a few to two hundred. These can be represented in the genetic construct as a single variant (one construct, one gene signature) or combined into a concatemer (several identified gene signatures arranged sequentially).
  • a mouse melanoma model was used to demonstrate the feasibility of the approach.
  • this example of using a bioinformatics algorithm to select gene signatures characteristic of tumor cells is universal and can be applied to different tumor types and different mammalian species, including humans.
  • mice female, 6-8 weeks old, average weight 20 g were used in the experiment. All animal studies were conducted in accordance with ethical standards.
  • Syngeneic murine melanoma B16-F10 cells were obtained from the collection of the Gamaleya National Research Center for Epidemiology and Microbiology of the Russian Ministry of Health. The cells were cultured in DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA), 100 U/ml penicillin and 100 ⁇ g/ml streptomycin (Gibco, USA) in an incubator with 5% CO2 at 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes.
  • Example 2 Creation of a DNA template for obtaining mRNA.
  • the DNA template is a circular plasmid DNA containing an open reading frame encoding the identified gene signatures.
  • DNA template variants were developed that contained both individual gene signatures of tumor cells and multiple gene signatures in a single reading frame.
  • the DNA template contains all the structural components necessary for efficient mRNA production in vitro, as well as elements necessary for DNA replication in E. coli and the ampicillin resistance gene.
  • mRNA After transcription from the designed DNA template, mRNA is formed, which has the sequence SEQ ID N0:2 before the open reading frame and SEQ ID N0:7 after the open reading frame.
  • sequences encoding the identified gene signatures (SEQ ID N0:9, SEQ ID N0:10, SEQ ID N0:11, SEQ ID N0:12, SEQ ID N0:13, SEQ ID N0:14, SEQ ID N0:15, SEQ ID N0:16, SEQ ID N0:17, SEQ ID N0:18) were synthesized from oligonucleotide primers and flanked by regions of homology with the plasmid vector required for the assembly of the DNA template.
  • the plasmid vector linearized at the Hindlll site and the PCR product (containing the gene signatures) were combined by the Gibson assembly method using the commercial Gibson Assembly® Ultra kit (Codex, USA). All molecular biology work (cloning) was performed using E. coli Top 10 electrocompetent cells.
  • the selection of oligonucleotide primers was carried out using the SnapGene v6.1.2 program.
  • Nucleic acid amplification was performed using MiniAmp (Thermofisher) and T100 (Biorad) instruments. Highly specific amplification of DNA fragments was performed using the 2X Platinum SuperFi Green MasterMix kit. Amplification conditions were as recommended by the manufacturer. PCR products for cloning from agarose gel were purified using the QIAquick Gel Extraction kit (QIAGEN) according to the manufacturer's instructions.
  • sequence encoding several gene signatures in a single reading frame (SEQ ID NO:19) and a sequence encoding three gene signatures in a single reading frame, connected via a linker (SEQ ID NO:20) were obtained.
  • SEQ ID NO:19 a sequence encoding several gene signatures in a single reading frame
  • SEQ ID NO:20 a sequence encoding three gene signatures in a single reading frame, connected via a linker
  • E. coli cells transformed with the DNA template were grown in 2xYT liquid medium (1.6% Tryptone, 1% Yeast extract, 0.5% NaCl) or on 2xYT solid medium + 2% agar with antibiotic. Plasmid DNA was isolated from a 4 ml overnight E. coli culture using the QIAGEN Plasmid Midi Kit (100) (QIAGEN: 12143) or QIAGEN Plasmid Maxi Kit (25) (QIAGEN: 12163) according to the standard protocol suggested by the manufacturer.
  • plasmid DNA isolation After plasmid DNA isolation, its concentration was measured on a Qubit®4.0 fluorimeter (Invitrogene, USA) using reagents from the commercial Qubit®dsDNA High Sensitivity Assay Kits (Life Technologies: Q32854) using the standard protocol suggested by the manufacturer. The correct assembly of the final plasmids was confirmed by Sanger sequencing on a Genetic Analyzer 3500 (Applied Biosystems) using the commercial BigDye® Terminator v3.1 Cycle Sequencing kit, according to the manufacturer's recommendations.
  • DNA matrices that contain an open reading frame with a luciferase sequence and encode various variants of mRNA structural elements, in particular:
  • a series of DNA matrices encoding mRNA with the sequence SEQ ID N0:2 up to the open reading frame and sequence SEQ ID N0:7 after the open reading frame wherein the reading frame encodes one or more tumor cell gene signatures (SEQ ID N0:9, or SEQ ID N0:10, or SEQ ID N0:11, or SEQ ID N0:12, or SEQ ID N0:13, or SEQ ID N0:14, or SEQ ID N0:15, or SEQ ID N0:16, or SEQ ID N0:17, or SEQ ID N0:18, or SEQ ID N0:19, or SEQ ID N0:20).
  • tumor cell gene signatures SEQ ID N0:9, or SEQ ID N0:10, or SEQ ID N0:11, or SEQ ID N0:12, or SEQ ID N0:13, or SEQ ID N0:14, or SEQ ID N0:15, or SEQ ID N0:16, or SEQ ID N0:17
  • DNA templates encoding mRNA with different structural elements at the 5’ (SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:8) and 3’ ends (SEQ ID N0:7, SEQ ID N0:7), with an open reading frame encoding luciferase, were obtained.
  • Example 3 Synthesis of mRNA based on the obtained DNA templates containing an open reading frame encoding the detected gene signatures.
  • ATP Addenosine - 5'-triphosphate
  • GTP Guanosine - 5'-triphosphate
  • STP Cytidine - 5'-triphosphate
  • N1-methylpseudouridine-5'-triphosphate Nl-Me-PseudoUTP
  • Table 1 shows the reaction mixture composition for a 100 ⁇ L reaction with 2 to 5 ⁇ g of DNA template. If necessary, the reaction volume can be reduced to 25 ⁇ L by sequentially decreasing the amounts of components. Before starting the IVT, all components are thawed on ice and vortexed. Next, the reaction is prepared, and the components are added in the order presented.
  • the reaction mixture is incubated at a temperature of +37°C for 120 minutes, after which 1 ⁇ l of DNase + 12 ⁇ l of DNase buffer (10x) (Synthol) are added to it and the mixture is incubated for another 30 minutes.
  • a set of mRNA with the sequence SEQ ID N0:2 before the open reading frame and the sequence SEQ ID N0:7 after the open reading frame was obtained, wherein the reading frame encodes one or more gene signatures of tumor cells (SEQ ID N0:9, or SEQ ID N0: 10, or SEQ ID N0:11, or SEQ ID N0:12, or SEQ ID N0: 13, or SEQ ID N0:14, or SEQ ID N0:15, or SEQ ID N0:16, or SEQ ID N0:17, or SEQ ID N0:18, or SEQ ID N0:19, or SEQ ID N0:20).
  • SEQ ID N0:9, or SEQ ID N0: 10 or SEQ ID N0:11, or SEQ ID N0:12, or SEQ ID N0: 13, or SEQ ID N0:14, or SEQ ID N0:15, or SEQ ID N0:16, or SEQ ID N0:17, or SEQ ID N0:18, or SEQ
  • a set of mRNAs with different structural elements at the 5’ (SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:8) and 3’ ends (SEQ ID N0:7, SEQ ID N0:7) with an open reading frame encoding luciferase were obtained.
  • Example 4 Preparation of lipid nanoparticles containing synthesized mRNA.
  • the mRNAs synthesized in Example 3 were encapsulated in lipid nanoparticles (LNPs) using a microfluidic mixing process of a rapid mRNA solution (pH 3.0) with a lipid solution dissolved in alcohol.
  • LNPs lipid nanoparticles
  • the lipids were dissolved in 96% ethanol at molar ratios of 46.3:9:42.7:1.6 (ionizable lipid: distearoylphosphatidylcholine (DSPC): cholesterol: PEGylated lipid (PEG lipid)).
  • DSPC distearoylphosphatidylcholine
  • PEG lipid PEGylated lipid
  • Acuitas ionizable lipid (ALC-0315) and PEG lipid (1,2-dimyristoyl-3-p-glycero-3-methoxypolyethyleneglycol 2000) were purchased from Cayman Chemical Company.
  • An mRNA solution with a working concentration of 0.2 mg/ml was prepared by mixing water Molecular biology grade, 10x citrate buffer (pH 3.0), and mRNA stock solution.
  • the lipid solution was combined with the mRNA solution (0.2 mg/mL) in a 3:1 volume ratio (aqueous solution: alcohol solution) using microfluidic mixing in a Nanoassmblr Benchtop system (Precision NanoSystems).
  • the ratio of ionizable nitrogen atoms in the ionizable lipid to the number of phosphate groups in mRNA (N:P ratio) was 6 for each composition.
  • the resulting formulations were dialyzed against PBS (pH 7.2) in 20 kDa Slide-A-Lyzer dialysis cassettes (Thermo Fisher Scientific) overnight with gentle stirring of the buffer solution on a magnetic stirrer in a refrigerator at +4°C.
  • the lipid nanoparticle suspension was withdrawn from the dialysis cassette using a syringe, filtered through a 0.2 ⁇ m Acrodisk filter (Supor membrane, Pall corporation), and the LNP preparations were stored at +4 °C until use.
  • a set of lipid particles was obtained containing mRNA with the sequence SEQ ID N0:2 before the open reading frame and the sequence SEQ ID N0:7 after the open reading frame, wherein the reading frame encodes one or more gene signatures of tumor cells (SEQ ID N0:9, or SEQ ID N0:10, or SEQ ID N0:11, or SEQ ID N0:12, or SEQ ID N0:13, or SEQ ID N0:14, or SEQ ID N0:15, or SEQ ID N0:16, or SEQ ID N0:17, or SEQ ID N0:18, or SEQ ID N0:19, or SEQ ID N0:20).
  • lipid particles containing mRNA with different structural elements at the 5’ (SEQ ID N0:1, SEQ ID N0:2, SEQ ID N0:3, SEQ ID N0:4, SEQ ID N0:5 SEQ ID N0:8) and 3’ ends (SEQ ID N0:7, SEQ ID N0:7), with an open reading frame encoding luciferase, were obtained.
  • the resulting lipid particles were used to deliver mRNA into mammalian cells.
  • Example 5 Evaluation of target antigen expression after introduction of various mRNA constructs encoding luciferase.
  • mice weighing approximately 18 g were used. The animals were divided into several experimental groups, which were administered:
  • mRNA that has the sequence SEQ ID N0:1 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end 2) mRNA that has the sequence SEQ ID N0:2 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:3 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:4 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:5 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:8 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:6 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:1 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:2 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:3 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end.
  • mRNA that has the sequence SEQ ID N0:4 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end.
  • mice were intraperitoneally injected with 100 ⁇ l of D-luciferin solution in PBS (25 mg/ml). Five minutes after injection, the animals were anesthetized with 1-2% isoflurane and placed in the IVIS L mina III imaging system (Perkin Elmer). Mice were imaged using Living Image software (Perkin Elmer).
  • Example 6 A method for using the developed immunobiological agent to induce an immune response to tumor cells containing gene signatures included in the mRNA vector.
  • the B16-F10 syngeneic melanoma cell line from mice C57N/6 was used in the experiment.
  • the cells were cultured in 75 cm2 culture flasks in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in an incubator containing 5% CO2 at a temperature of 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes. They were then pelleted in a centrifuge at 1000 rpm for 10 minutes.
  • phosphate-buffered saline was added to the pellet, mixed, and pelleted in a centrifuge at 1000 rpm for 10 minutes. Washing with phosphate-buffered saline was performed twice. Cells were diluted in 1 ⁇ phosphate-buffered saline at a concentration of 2x10 6 cells per milliliter.
  • mice were anesthetized with 3% isoflurane.
  • Cells were injected subcutaneously into the right flank of the mouse at 100 ⁇ l (2 x 105 cells per mouse) using a 1 ml syringe with a 22G needle.
  • the animals were divided into several groups, depending on the type of drug they were administered:
  • the study drug was administered the following day after tumor cell inoculation at a dose of 20 ⁇ g mRNA per mouse using an insulin syringe with a 29G needle.
  • the drug was administered intramuscularly every 4 days.
  • tumor size was determined and its volume calculated.
  • the data obtained are presented in Figure 2.
  • the introduction of mRNA with an open reading frame encoding the detected tumor cell gene signatures results in tumor cell growth restriction. This establishes an immune surveillance system for tumor cells in mammals, enabling effective detection and elimination of tumor cells.
  • Example 7 A method for using the developed immunobiological agent to induce an immune response to tumor cells, in which more than 2 immunobiological agents encoding different tumor gene signatures are simultaneously administered.
  • the B16-F10 syngeneic melanoma cell line from mice C57 ⁇ /6 was used in the experiment.
  • the cells were cultured in 75 cm2 culture flasks in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in an incubator with 5% CO2 at a temperature of 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes.
  • DMEM Dulbecco's Modified Eagle's Medium
  • mice were anesthetized with 3% isoflurane.
  • Cells were injected subcutaneously into the right flank of the mouse at 100 ⁇ l (2 x 105 cells per mouse) using a 1 ml syringe with a 22G needle.
  • the animals were divided into several groups, which were administered:
  • the study drug was administered the following day after tumor cell inoculation at a dose of 20 ⁇ g mRNA (the total amount was divided proportionally by the amount of mRNA vectors) per mouse using an insulin syringe with a 29G needle.
  • the drug was administered intramuscularly every 4 days.
  • tumor size was determined and its volume was calculated.
  • the data obtained are presented in Fig. 3.
  • the introduction of several open reading frame mRNA variants encoding the detected gene signatures of tumor cells leads to growth restriction. Tumor cells. This creates an immune surveillance system for tumor cells in mammals, which allows for the effective detection and elimination of tumor cells.
  • Example 8 Changes in the tumor microenvironment.
  • the B16-F10 syngeneic melanoma cell line from mice C57 ⁇ /6 was used in the experiment.
  • the cells were cultured in 75 cm2 culture flasks in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% fetal bovine serum, penicillin, streptomycin, and L-glutamine in an incubator with 5% CO2 at a temperature of 37°C.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the cells were detached with trypsin and transferred to centrifuge tubes. They were then pelleted in a centrifuge at 1000 rpm for 10 minutes.
  • phosphate-buffered saline was added to the pellet, mixed, and pelleted in a centrifuge at 1000 rpm for 10 minutes. Washing with phosphate-buffered saline was performed twice. Cells were diluted in 1 ⁇ phosphate-buffered saline at a concentration of 2x10 6 cells per milliliter.
  • mice were anesthetized with 3% isoflurane.
  • Cells were injected subcutaneously into the right flank of the mouse at 100 ⁇ l (2 x 105 cells per mouse) using a 1 ml syringe with a 22G needle.
  • the animals were divided into 2 groups, which were administered every 4 days:
  • the drug administration started on day 1 of the study.
  • mice intact animals
  • mice injected with the study drug were euthanized with carbon dioxide.
  • the tumor was separated from the surrounding tissue with scissors, then minced and passed through a 100- ⁇ m nylon strainer in 10 ml of phosphate-buffered saline containing 1% fetal bovine serum.
  • the cell suspension was pelleted at 450g for 10 minutes, and the pellet was resuspended in 1 ml of phosphate-buffered saline containing 1% fetal bovine serum.
  • 1 million cells were collected from the suspension and pelleted at 450g for 10 minutes. Fc-block was added to the pellet, and the mixture was incubated for 30 minutes at +4°C.
  • phosphate-buffered saline 1 ml was added to the cell suspension, and the mixture was pelleted at 450g for 10 minutes. A mixture of antibodies diluted in staining buffer (BD) was added to the sediment. Antibodies to the following cell markers were used in the study: CD45 - a marker of all leukocytes, CD3 - a marker of T-lymphocytes, F4/80 - a marker of macrophages. Then, to the suspension 1 ml of phosphate-buffered saline was added to each cell and pelleted at 450 g for 10 minutes. The pellet was resuspended in 100 ⁇ l of phosphate-buffered saline with DAPI and analyzed by flow cytometry. DAPI was used to assess cell viability, as this dye only penetrates cells with damaged membranes.
  • staining buffer BD
  • the obtained data show that the developed immunobiological agent induces immune responses that alter the tumor microenvironment and initiates the detection of tumor cells by the immune system, as well as their elimination.

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Abstract

Ce groupe d'inventions se rapporte au domaine de l'immunologie et de l'oncologie. Cet agent immunobiologique peut être utilisé dans le traitement de patients soufrant de maladies oncologiques d'étiologies différentes. L'invention concerne un agent à base d'un vecteur ARNm, dans lequel l'ARNm comprend une séquence SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8 à la 5' extrémité; un cadre de réticulation ouvert codant de 1 à 80 signatures gènes tumoraux; et une séquence SEQ ID NO:6 ou SEQ ID NO:7 à la 3' extrémité. L'invention concerne également un procédé de production de l'agent et son utilisation. Ce groupe d'inventions permet d'élargir l'éventail des moyens et des procédés pour induire une réponse immune chez des patients soufrant de maladies oncologiques.
PCT/RU2025/000257 2024-06-29 2025-08-28 Agent immunobiologique pour traiter des maladies oncologiques sur la base d'un vecteur arn Pending WO2026005647A2 (fr)

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