Method for creating a tumour cell immunosurveillance system in a mammalian organism

Abstract

The invention relates to immunology and oncology. The proposed invention can be used in the treatment of patients with cancerous diseases of various origin. What is proposed is a method for creating a tumour cell immunosurveillance system in a mammalian organism which includes a) identifying differences in the gene signatures of tumour cells and normal cells, b) synthesizing a set of DNA matrices, each of which contains an open reading frame encoding one of the detected gene signatures, or an open reading frame encoding 2 to 80 detected gene signatures, c) synthesizing a set of mRNA from the obtained set of DNA matrices, d) producing lipid nanoparticles containing the synthesized mRNA, and e) introducing said lipid nanoparticles into a mammalian organism. Creating a tumour cell immunosurveillance system in mammals enables the immune system to efficiently detect and destroy tumour cells.

Description

[0001] A method for forming an immune surveillance system for tumor cells in the body of mammals.

[0002] Field of technology

[0003] The invention relates to immunology and oncology. It can be used in the treatment of patients with cancer of various etiologies. The development of an immune surveillance system for tumor cells in mammals allows the immune system to effectively detect and destroy tumor cells. Furthermore, the present invention can be used to regulate the tumor microenvironment.

[0004] State of the art

[0005] 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].

[0006] According to the International Agency for Research on Cancer (IARC), the number of cancer cases worldwide could increase by approximately 77% by 2050. [Cancer rates set to rise 77 percent by 2050 https: / / news.un.org / en / story / 2024 / 02 / 1146127. Accessed June 18, 2024]. The rapid increase in the global cancer burden is a consequence of both population aging and demographic growth, as well as changes in people's exposure to risk factors.

[0007] Cancer development involves the progressive transformation of normal cells into malignant ones, which results from genetic and epigenetic changes occurring within the cells. Initially, tumor cells can be eliminated by immune system effector cells. However, this process leads to immune selection (tumor immunoediting), which favors the selection of tumor cell clones with less immunogenicity and the ability to block the activity of immunocompetent cells. Ultimately, during tumor progression, when the tumor reaches a clinically detectable size, soluble factors secreted by tumor cells can create favorable conditions in the tumor microenvironment for immune evasion.

[0008] 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].

[0009] The discovery of immune checkpoint inhibitors has 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. Currently, four immune checkpoint inhibitors are registered in Russia: ipilimumab, nivolumab, pembrolizumab, and atezolizumab. Furthermore, this field is actively developing, and new targets are being sought. However, 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.

[0010] Another area of ​​advancement in oncoimmunology is the development of therapeutic vaccines. There are several classes of these drugs, each targeting different antigens and eliciting a cell-mediated immune response:

[0011] 1) Dendritic cell (DC)-based vaccines. 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 that present 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]. Cancer vaccines of this type typically include DC isolated from patients or obtained ex vivo by culturing the patient's hematopoietic progenitor cells or monocytes. DC are additionally loaded with tumor antigens and are sometimes combined with immunostimulatory agents such as GM-CSF.Although the collected data indicate that DC-based vaccines are well tolerated and have a good safety profile, clear therapeutic results are achieved in less than 15% of patients Calmeiro J, Carrascal MA, Tavares AR, Ferreira DA, Gomes C, Falcao A, Cruz MT, Neves BM. Dendritic Cell Vaccines for Cancer Immunotherapy: The Role of Human Conventional Type 1 Dendritic Cells. Pharmaceutics. 2020 Feb 15;12(2):158. doi: 10.3390 / pharmaceuticsl2020158. PMID: 32075343; PMCID: PMC7076373.

[0012] 2) 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, currently in clinical trials, are based on both autologous (e.g., OncoVAX, LipoNova) and allogeneic (e.g., Canvaxin, Onyvax-P, GV AX) tumor cell lines. 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. It has also been shown that immune recognition of tumor cells by CD8+ T cells and antigen-presenting cells is enhanced after irradiation.Furthermore, whole-cell vaccines can be modified to enhance immunogenicity by transfecting immunostimulatory molecules. For example, the GVAX vaccine platform utilizes irradiated allogeneic tumor cell lines modified to secrete GM-CSF to enhance the vaccine's immunogenicity. Despite these advantages, this type of vaccine has several drawbacks. For example, when cells are irradiated, phosphatidylserine, an immunosuppressive phospholipid normally found on the inner leaflet of the plasma membrane, is translocated from the inner to the outer surface of the plasma membrane. This, in turn, leads to the secretion of immunosuppressive factors by dendritic cells and inhibits their maturation, promoting an immunosuppressive environment for the tumor. Furthermore, irradiated cells may retain the ability to secrete immunosuppressive factors, similar to the original tumor cells.Thus, radiation-induced immune suppression partially negates the immunogenic effect of irradiated whole cells [Srivatsan S, Patel JM, Bozeman EN, Imasuen IE, He S, Daniels D, Selvaraj P. Allogeneic tumor cell vaccines: the promise and limitations in clinical trials. Hum Vaccin Immunother. 2014;10(l):52-63. doi: 10.4161 / hv.26568. Epub 2013 Sep 24. PMID: 24064957; PMCID: РМС4181031].

[0013] 3) 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). 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.However, although preclinical data have often shown encouraging results, clinical trials of therapeutic cancer vaccines, including peptide-based vaccines, have to date yielded insufficient data. The limited efficacy of peptide-based cancer vaccines is a consequence of several factors, including the identification of specific tumor antigen targets, the limited immunogenicity of the peptides, and the highly immunosuppressive tumor microenvironment.

[0014] Thus, there is a need in the art to develop methods for forming an immune surveillance system of the body that will allow the immune system to effectively recognize and eliminate tumor cells.

[0015] Disclosure of invention

[0016] The aim of the present invention is to develop a method for detecting tumor cells for the purpose of their destruction by the immune system in vivo.

[0017] The technical result consists in developing a method for forming an immune surveillance system for tumor cells in the body of mammals, which makes it possible to detect tumor cells and initiate an effective immune response against these cells. The said technical result is achieved by developing a method for forming an immune surveillance system for tumor cells in the body of mammals, which includes a) identifying differences in the gene signatures of tumor and normal cells; b) synthesizing a set of DNA matrices, each of which contains an open reading frame encoding one of the detected gene signatures or an open reading frame encoding from 2 to 80 detected gene signatures; c) synthesizing a set of mRNA based on the obtained set of DNA matrices; d) obtaining lipid nanoparticles containing the synthesized mRNA; d) introducing the obtained lipid nanoparticles into the body of mammals.

[0018] In one embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, wherein the mRNA has the sequence SEQ ID N0:1 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0019] In another embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, in which the mRNA has the sequence SEQ ID N0:2 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0020] A variant of the invention has also been developed in which the mRNA has the sequence SEQ ID N0:3 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0021] In addition, a variant of the invention has been developed in which the mRNA has the sequence SEQ ID N0:4 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0022] In another embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, in which the mRNA has the sequence SEQ ID N0:5 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0023] There is also a variant of the invention in which the mRNA has the sequence SEQ ID N0:8 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

[0024] In another embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, in which the mRNA has the sequence SEQ ID N0:1 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0025] In another embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, in which the mRNA has the sequence SEQ ID N0:2 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0026] A variant of the invention has also been developed in which the mRNA has the sequence SEQ ID N0:3 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0027] In addition, a variant of the invention has been developed in which the mRNA has the sequence SEQ ID N0:4 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0028] In another embodiment, the present invention describes a method for forming an immune surveillance system for tumor cells in a mammalian body, in which the mRNA has the sequence SEQ ID N0:5 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0029] There is also a variant of the invention in which the mRNA has the sequence SEQ ID N0:8 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

[0030] In a particular embodiment of the present invention, the method for forming an immune surveillance system is characterized by a change in the tumor microenvironment.

[0031] Implementation of the invention

[0032] Brief description of the drawings. Fig. 1 shows the results of in vivo bioluminescence measurements using the IVIS Imaging System (Perkin Elmer, USA).

[0033] The numbers in the photograph indicate animals that were injected with the following mRNA variants:

[0034] 1. 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.

[0035] 2. 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.

[0036] 3. 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.

[0037] 4. 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.

[0038] 5. 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.

[0039] 6. 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.

[0040] 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.

[0041] 8. 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.

[0042] 9. 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.

[0043] 10. 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.

[0044] 11. 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.

[0045] 12. 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.

[0046] Fig. 2 shows the results of measuring the tumor volume in animals that were administered a phosphate-buffered solution and the studied mRNA preparations.

[0047] The ordinate axis shows the tumor volume, mm 3 .

[0048] The abscissa axis shows different groups of animals, where

[0049] 1. Control mice injected with phosphate-buffered saline 2. Mice injected with mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:9;

[0050] 3. Mice injected with mRNA containing an open reading frame encoding the detected gene signature SEQ ID N0:10;

[0051] 4. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID NO: 11;

[0052] 5. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:12;

[0053] 6. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:13;

[0054] 7. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:14;

[0055] 8. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:15;

[0056] 9. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:16;

[0057] 10. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:17;

[0058] 11. Mice injected with mRNA containing an open reading frame encoding the identified gene signature SEQ ID N0:18;

[0059] 12. Mice injected with mRNA containing an open reading frame encoding the 10 identified gene signatures of SEQ ID N0:19;

[0060] 13. Mice injected with mRNA containing an open reading frame encoding the 3 identified gene signatures of SEQ ID N0:20.

[0061] Fig. 3 shows the results of measuring the tumor volume in animals that were administered a phosphate-buffered solution and the studied mRNA preparations.

[0062] The ordinate axis shows the tumor volume, mm 3 .

[0063] The abscissa axis shows different groups of animals, where

[0064] 1. Phosphate buffered saline

[0065] 2. mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:9;

[0066] 3. mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:10; 4. mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:10 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:11;

[0067] Fig. 4 shows the results of determining the cellular composition of the tumor microenvironment, where

[0068] A - assessment of the percentage of macrophages,

[0069] B - T-lymphocytes.

[0070] 1 - intact mice (no drug was administered), 2 - the drug under study.

[0071] * - p<0.05, Mann-Whitney test.

[0072] In response to stress, 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.

[0073] The developed method involves sequencing DNA isolated from a patient's tumor cells, which results in 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 that are strictly specific to tumor cells. A set of DNA matrices containing an open reading frame encoding the identified gene signatures is then created. One variant is possible in which a single DNA matrix contains an open reading frame encoding a single identified gene signature. Alternatively, 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 in this variant 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. In addition to the open reading frame, 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.

[0074] Several variants of DNA matrices have been developed encoding:

[0075] 1) 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.

[0076] 2) 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.

[0077] 3) 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.

[0078] 4) 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.

[0079] 5) 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.

[0080] 6) 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.

[0081] 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.

[0082] 8) 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.

[0083] 9) 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.

[0084] 10) 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.

[0085] 11) 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.

[0086] 12) mRNA with the sequence SEQ ID N0:8 upstream of the open reading frame and the sequence SEQ ID N0:7 downstream of the open reading frame. In all presented mRNA sequences, uridine was replaced with pseudouridine.

[0087] Example 5 demonstrates that all of the listed mRNA constructs are capable of providing mRNA translation. For this purpose, 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. Thus, after introducing these mRNAs into mice, it was possible to assess luciferase synthesis by a reaction with luciferin, which is accompanied by luminescence. In addition, the authors obtained various versions of these constructs with an open reading frame encoding tumor gene signatures that exhibited high antitumor activity. mRNAs obtained by in vitro transcription using the developed DNA templates were subsequently packaged into lipid nanoparticles and administered into mammals.

[0088] Translation of mRNA containing an open reading frame encoding tumor cell-specific gene signatures has been shown to facilitate immune system recognition of tumor antigens. As a result, the immune system effectively targets and destroys tumor cells, thereby establishing a tumor surveillance system. This effect has been shown to correlate with changes in the tumor microenvironment. The invention is supported by the following examples.

[0089] Example 1. Identification of differences in the gene signatures of tumor and normal cells.

[0090] 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.Depending on the tumor's genetic landscape, 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. However, 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.

[0091] C57BL / 6 mice (female, 6-8 weeks old, average weight 20 g) were used in the experiment. All animal studies complied with ethical standards. Syngeneic B16-F10 murine melanoma 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 μg / ml streptomycin (Gibco, USA) in an incubator containing 5% CO2 at 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes. They were then centrifuged at 1000 rpm for 10 minutes. Next, 1× phosphate-buffered saline was added to the pellet, mixed, and centrifuged at 1000 rpm for 10 minutes. The cells were washed twice with phosphate-buffered saline. The cells were diluted in 1× phosphate-buffered saline at a concentration of 2x10 6cells / ml. Before the introduction of tumor cells, C57b / 6 mice were anesthetized with inhalation anesthesia (3% isoflurane). The cells were injected subcutaneously into the right side of the mouse at 100 μl (2x10 5 cells per mouse).

[0092] After 12 days, tumor and healthy tissue samples were collected. Whole-genome, exome, and transcriptome sequencing were then performed on an MGI G400 (MGI, China). The resulting data were analyzed using proprietary software, which identified approximately 500 mutations characteristic of this tumor. Based on the resulting mutanomes, a list of specific antigens was generated, which was then filtered and prioritized according to several criteria: mutant gene expression level, uniqueness (to minimize the risk of autoimmune reactions), and aggretopicity. Thus, as a result of the work carried out, 10 gene signatures characteristic of this type of tumor were identified: 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. The obtained sequences were then used to obtain DNA matrices.

[0093] Example 2. Creation of a DNA template for obtaining mRNA.

[0094] The goal of this stage of the work was to create a DNA template for mRNA production. The DNA template is a circular plasmid DNA containing an open reading frame encoding the identified gene signatures. During the course of the work, DNA template variants were developed that contained both individual gene signatures of tumor cells and multiple gene signatures in a single reading frame.

[0095] In addition to the open 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.

[0096] 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.

[0097] The 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. Next, 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.

[0098] 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.

[0099] In addition, a 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. These sequences were synthesized by Evrogen JSC and inserted into a DNA matrix using genetic engineering methods similar to those described above.

[0100] 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 antibiotics. 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. 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) according to 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.

[0101] In addition, a series of DNA matrices were obtained that contain an open reading frame with a luciferase sequence and encode various variants of mRNA structural elements, in particular:

[0102] 13) 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.

[0103] 14) 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.

[0104] 15) 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.

[0105] 16) 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.

[0106] 17) 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.

[0107] 18) 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. 19) 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.

[0108] 20) 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.

[0109] 21) 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.

[0110] 22) 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.

[0111] 23) 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.

[0112] 24) 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.

[0113] Thus, as a result of the work carried out, a series of DNA matrices were obtained encoding 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).

[0114] In addition, 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.

[0115] Example 3. Synthesis of mRNA based on the obtained DNA templates containing an open reading frame encoding the detected gene signatures.

[0116] To synthesize mRNA, an in vitro transcription reaction was performed using DNA templates obtained in the previous example.

[0117] To carry out the in vitro transcription reaction, the following components are required:

[0118] - a mixture of nucleotides (Adenosine - 5'-triphosphate (ATP), Guanosine - 5'-triphosphate (GTP), Cytidine - 5'-triphosphate (STP) and M-methylpseudouridine-5'-triphosphate (Nl-Me-PseudoUTP)) (Biolabmix);

[0119] - analog of the cap structure! m7GmAmG (Biolabmix); - RNase inhibitor (Biolabmix);

[0120] - pyrophosphatase;

[0121] - T7 RNA polymerase (Biolabmix);

[0122] - T7 buffer for IVT;

[0123] - additional buffer.

[0124] Table 1 shows the reaction mixture composition for a 100 µL reaction with 2 to 5 µg of DNA template. If necessary, the reaction volume can be reduced to 25 µL 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.

[0125] Table 1. Composition of the reaction for the synthesis of mRNA with cotranscriptional capping by the cap-1 analog m7GmAmG

[0126] The reaction mixture is incubated at a temperature of +37°C for 120 minutes, after which 1 µl of DNase + 12 µl of DNase buffer (10x) (Synthol) are added to it and the mixture is incubated for another 30 minutes.

[0127] Thus, as a result of the work carried out, 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).

[0128] In addition, a set of mRNAs with different 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 were obtained.

[0129] 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. For this purpose, 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)). 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 molecular biology grade water, 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 on 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 a PBS solution (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. Upon completion of dialysis, the lipid nanoparticle suspension was withdrawn from the dialysis cassette with a syringe, filtered through a 0.2 μm Acrodisk filter (Supor membrane, Pall corporation), and stored at +4°C until use.

[0130] Thus, as a result of the work carried out, 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 NO: 18, or SEQ ID NO: 19, or SEQ ID N0:20).

[0131] In addition, a set of 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.

[0132] The resulting lipid particles were used to deliver mRNA into mammalian cells.

[0133] Example 5. Evaluation of target antigen expression after administration of various mRNA constructs encoding luciferase.

[0134] In this experiment, BALB / c mice weighing approximately 18 g were used. The animals were divided into several experimental groups, which were administered:

[0135] 1) 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.

[0136] 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.

[0137] 3) 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.

[0138] 4) 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.

[0139] 5) 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.

[0140] 6) 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.

[0141] 7) 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.

[0142] 8) an mRNA that has the sequence of SEQ ID NO:2 at the 5' end, an open reading frame encoding luciferase and the sequence of SEQ ID NO:7 at the 3' end. 9) an mRNA that has the sequence of SEQ ID NO:3 at the 5' end, an open reading frame encoding luciferase and the sequence of SEQ ID NO:7 at the 3' end.

[0143] 10) 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.

[0144] 11) mRNA that has the sequence SEQ ID N0:5 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end.

[0145] 12) mRNA that has the sequence SEQ ID N0:8 at the 5' end, an open reading frame encoding luciferase and the sequence SEQ ID N0:7 at the 3' end

[0146] Three hours after administration of the experimental agents, mice were intraperitoneally injected with 100 μl of a 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 Lumina III imaging system (Perkin Elmer). Mice were imaged using Living Image software (Perkin Elmer).

[0147] The obtained data are shown in Fig. 1. As can be seen from the results of the experiment, after the introduction of all the studied mRNA constructs, expression of luciferase was observed at the injection site and in the liver of the animals.

[0148] 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.

[0149] The experiment used the B16-F10 cell line, a syngeneic melanoma cell line from mice C57Ы / 6. The cells were cultured in 75 cm 2Cells were cultured in 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 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes. They were then centrifuged at 1000 rpm for 10 minutes. The precipitate was then added with 1× phosphate-buffered saline, mixed, and centrifuged at 1000 rpm for 10 minutes. The mixture was washed twice with phosphate-buffered saline. Cells were diluted in 1× phosphate-buffered saline at a concentration of 2x10 6 cells per milliliter.

[0150] Before the introduction of tumor cells, C57N / 6 mice were anesthetized with inhalation anesthesia (3% Isoflurane). The cells were injected subcutaneously into the right side of the mouse at 100 μl (2x10 5 cells per mouse) with a 1 ml syringe with a 22G needle.

[0151] The animals were divided into several groups, depending on the type of drug they were administered:

[0152] 1) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:9;

[0153] 2) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:10;

[0154] 3) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:11;

[0155] 4) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:12;

[0156] 5) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:13;

[0157] 6) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:14;

[0158] 7) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:15;

[0159] 8) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:16;

[0160] 9) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:17;

[0161] 10) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:18;

[0162] 11) mRNA with an open reading frame encoding 10 detected gene signatures SEQ ID N0:19;

[0163] 12) mRNA with an open reading frame encoding 3 detected gene signatures SEQ ID N0:20.

[0164] 13) Phosphate-buffered saline. The test drug was administered the following day after tumor cell inoculation at a dose of 20 μg mRNA per mouse using an insulin syringe with a 29G needle. The drug was administered intramuscularly every 4 days.

[0165] On day 8 of the study, tumor size was determined and its volume calculated. The data obtained are presented in Figure 2. As can be seen from the experimental results, 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.

[0166] 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.

[0167] The experiment used the B16-F10 cell line, a syngeneic melanoma cell line from mice C57Ы / 6. The cells were cultured in 75 cm 2Cells were cultured in 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 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes. They were then centrifuged at 1000 rpm for 10 minutes. The precipitate was then added with 1× phosphate-buffered saline, mixed, and centrifuged at 1000 rpm for 10 minutes. The mixture was washed twice with phosphate-buffered saline. Cells were diluted in 1× phosphate-buffered saline at a concentration of 2x10 6 cells per milliliter.

[0168] Before the introduction of tumor cells, C57N / 6 mice were anesthetized with inhalation anesthesia (3% Isoflurane). The cells were injected subcutaneously into the right side of the mouse at 100 μl (2x10 5 cells per mouse) with a 1 ml syringe with a 22G needle.

[0169] The animals were divided into several groups, which were administered:

[0170] 1) mRNA with an open reading frame encoding the detected gene signature SEQ ID N0:9 once;

[0171] 2) mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:10; 3) mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:10 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:9 and mRNA with an open reading frame encoding the detected gene signature of SEQ ID N0:11;

[0172] 4) Phosphate buffered saline

[0173] The study drug was administered the following day after tumor cell inoculation at a dose of 20 μg 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.

[0174] On day 8 of the study, tumor size was determined and its volume was calculated. The data obtained are presented in Figure 3. As can be seen from the experimental results, the introduction of several open reading frame mRNA variants 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.

[0175] Example 8. Changes in the tumor microenvironment.

[0176] The experiment used the B16-F10 cell line, a syngeneic melanoma cell line from mice C57Ы / 6. The cells were cultured in 75 cm 2 Cells were cultured in 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 37°C. Upon reaching 70-80% confluency, the cells were detached with trypsin and transferred to centrifuge tubes. They were then centrifuged at 1000 rpm for 10 minutes. The precipitate was then added with 1× phosphate-buffered saline, mixed, and centrifuged at 1000 rpm for 10 minutes. The mixture was washed twice with phosphate-buffered saline. Cells were diluted in 1× phosphate-buffered saline at a concentration of 2x10 6 cells per milliliter.

[0177] Before the introduction of tumor cells, C57N / 6 mice were anesthetized with inhalation anesthesia (3% Isoflurane). The cells were injected subcutaneously into the right side of the mouse at 100 μl (2x10 5cells per mouse) with a 1 ml syringe with a 22G needle.

[0178] Animals were divided into 2 groups, which were administered every 4 days: 1) mRNA with an open reading frame encoding 21 detected gene signatures SEQ ID N0:19.

[0179] 2) Phosphate buffered saline

[0180] The drug administration started on day 1 of the study.

[0181] Control mice (intact animals) and 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-μm 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. Next, 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. Then, 1 ml of phosphate-buffered saline 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 for all leukocytes), CD3 (a marker for T lymphocytes), and F4 / 80 (a marker for macrophages).Next, 1 ml of phosphate-buffered saline was added to the cell suspension and pelleted at 450 g for 10 minutes. The pellet was resuspended in 100 µl 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.

[0182] The obtained data are presented in Figure 4. As can be seen from the experimental results, after administration of mRNA containing the open reading frame encoding the detected gene signature, the tumor microenvironment changes. Specifically, the number of F4 / 80+ macrophages, which play a key role in tumor vascularization, decreases, while the number of CD3+ T lymphocytes increases, which, according to literature data, is a prognostic marker for successful therapy.

[0183] Thus, the obtained data show that the developed method for forming an immune surveillance system for tumor cells in the body of mammals leads to changes in the microenvironment of tumor cells.

[0184] Industrial applicability.

[0185] The developed method for forming an immune surveillance system for tumor cells in the body of mammals can be used to treat patients with oncological diseases of various etiologies.

Claims

Invention formula 1. A method for forming an immune surveillance system for tumor cells in a mammalian body, comprising a) identifying differences in the gene signatures of tumor and normal cells, b) synthesizing a set of DNA matrices, each of which contains an open reading frame encoding one of the detected gene signatures or an open reading frame encoding from 2 to 80 detected gene signatures; c) synthesizing a set of mRNA based on the obtained set of DNA matrices; d) obtaining lipid nanoparticles containing the synthesized mRNA; c) introducing the obtained lipid nanoparticles into the mammalian body.

2. The method according to claim 1, wherein the mRNA has the sequence SEQ ID N0:1 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

3. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:2 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:6 after the open reading frame encoding the detected gene signatures.

4. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:3 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:6 after the open reading frame encoding the detected gene signatures.

5. The method according to claim 1, wherein the mRNA has the sequence SEQ ID N0:4 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

6. The method according to claim 1, wherein the mRNA has the sequence SEQ ID N0:5 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:6 after the open reading frame encoding the detected gene signatures.

7. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:8 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:6 after the open reading frame encoding the detected gene signatures.

8. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:1 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:7 after the open reading frame encoding the detected gene signatures.

9. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:2 up to the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID N0:7 after the open reading frame encoding the detected gene signatures.

10. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:3 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:7 after the open reading frame encoding the detected gene signatures.

11. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:4 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:7 after the open reading frame encoding the detected gene signatures.

12. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:5 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:7 after the open reading frame encoding the detected gene signatures.

13. The method according to claim 1, wherein the mRNA has the sequence SEQ ID NO:8 before the open reading frame encoding the detected gene signatures, and has the sequence SEQ ID NO:7 after the open reading frame encoding the detected gene signatures.

14. A method for forming an immune surveillance system according to paragraph 1, which results in a change in the tumor microenvironment.

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