Application of GBP2 and / or GBP2 mRNA in preparation of medicine for treating tumors and medicine

Abstract

The invention relates to application of GBP2 and / or GBP2mRNA in preparation of a medicine for treating tumors and the medicine. Compared with other medicines in clinical experiments, the GBP2 has the obvious advantages that the GBP2 is a key molecule of an I FN-gamma signal channel and plays an important role in removing foreign pathogens, so that the tolerance of a patient to the GBP2 is high. The GBP2 can promote the formation of an anti-tumor immune microenvironment and kill tumor cells through an I FN-gamma pathway. GBP2 can inhibit the growth of melanoma and improve the survival rate; t cells are promoted to kill tumors, melanoma growth is inhibited, and the survival rate is increased; gBP2 is used for promoting infiltration of CD8 < + > T cells; gBP2 promotes formation of memory T cells; gBP2 promotes immune checkpoint expression of tumor cells; the GBP2 is used for promoting the generation of cell factors; the GBP2 is used for promoting the generation of a chemotactic factor CXCL9; and the GBP2 promotes the improvement of the immunogenic molecule HMGB1.

Description

Technical Field

[0001] The present application relates to the field of biomedical technology, and in particular to an application of GBP2 and / or GBP2 mRNA in the preparation of a drug for treating tumors and a drug. Background Art

[0002] Melanoma is a neoplasm formed when melanocytes lose normal regulation of their growth at the genetic level under the action of various carcinogenic factors, resulting in their clonal abnormal proliferation. It is common in the middle-aged and elderly population (the largest age range: 50-69 years old) and can be seen in various parts of the human body. There is a big difference in the classification of melanoma between China and the West. In the United States, melanoma is mainly skin-type, while in China, melanoma is mostly acral and mucosal. More than half of the melanoma patients in my country are in the middle and late stages (stages III-IV) at the time of initial diagnosis, and only 13% of patients are in the early stages (stages I-II). At the same time, in terms of pathological diagnosis, 66% of patients were clearly accompanied by ulcers, and the average infiltration depth was 3.81mm, which means that in addition to the late stage, the prognosis of melanoma patients in my country is also poor. At present, traditional tumor treatment methods such as surgical treatment, chemotherapy, radiotherapy, etc. cannot meet the clinical and patient needs due to a series of problems such as large side effects on the body, unstable treatment effects, and the risk of recurrence.

[0003] Tumor immunotherapy refers to a treatment method that stimulates or rebuilds the body's immune system in an active or passive way, so that the body produces a tumor-specific immune response, thereby controlling and killing tumor cells. It has the advantages of high efficiency, specificity, few side effects, and high safety. Unlike traditional treatment methods such as surgery, chemotherapy, and radiotherapy, tumor immunotherapy reshapes the tumor immune microenvironment and activates the body's anti-tumor immune response to achieve the effect of tumor clearance. In recent years, immune checkpoint blockade therapy (ICB) represented by PD-1 / PD-L1 antibodies has achieved remarkable clinical efficacy in the treatment of nearly 20 solid tumors including lung cancer, melanoma, gastrointestinal tumors, breast cancer, urinary system tumors, skin cancer, lymphoma, etc., becoming a milestone breakthrough in the history of tumor treatment. Adoptive T cell therapy is an immunotherapy that uses the patient's own immune cells to detect and eliminate tumor cells. It uses the patient's own (autologous transplantation) or donor (allogeneic transplantation) immune cells to improve immune function. However, some cancer patients show treatment resistance during the initial treatment. The objective response rate of melanoma patients is only 20-50%. Some patients who achieve good therapeutic effects in the early stage will still experience recurrence and progression, and only a handful of patients can maintain long-term benefits.

[0004] In recent years, the potential of RNA vaccines in cancer treatment has attracted widespread attention. Among them, mRNA vaccines have attracted widespread attention due to their rapid development and high customization, and their successful application in vaccine research and development has quickly turned to cancer treatment research.

[0005] Therefore, overcoming the "drug resistance" of melanoma immunotherapy and finding other new and effective drugs are key issues that need to be urgently addressed in melanoma immunotherapy. Summary of the invention

[0006] The embodiments of the present application provide an application of GBP2 and / or GBP2 mRNA in the preparation of a drug for treating tumors and the drug. GBP2 can promote the formation of an anti-tumor immune microenvironment and kill tumor cells through the IFN-γ pathway.

[0007] In a first aspect, the embodiments of the present application provide a use of GBP2 and / or GBP2 mRNA in the preparation of a drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy.

[0008] Furthermore, the gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1;

[0009] And / or, the amino acid sequence of GBP2 is shown as SEQ ID NO.2.

[0010] Furthermore, the drug includes lipid nanoparticles, and the lipid nanoparticles are encapsulated outside the GBP2 mRNA.

[0011] Furthermore, the tumor immunotherapy includes adoptive T cell therapy.

[0012] Furthermore, the tumor includes one or more of melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric tumors, cervical cancer, ovarian cancer, bladder cancer, urothelial carcinoma, ureteral tumors, prostate cancer, seminoma, testicular tumors, head and neck tumors, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

[0013] In a second aspect, the embodiments of the present application provide a drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy, which includes an active substance, and the active substance includes GBP2 and / or GBP2 mRNA.

[0014] Furthermore, the gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1;

[0015] And / or, the amino acid sequence of GBP2 is shown as SEQ ID NO.2.

[0016] Furthermore, it also includes lipid nanoparticles, and the lipid nanoparticles are wrapped outside the GBP2 mRNA.

[0017] Furthermore, the tumor immunotherapy includes adoptive T cell therapy.

[0018] Furthermore, the tumor includes one or more of melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric tumors, cervical cancer, ovarian cancer, bladder cancer, urothelial carcinoma, ureteral tumors, prostate cancer, seminoma, testicular tumors, head and neck tumors, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

[0019] The beneficial effects of the technical solution provided by this application include:

[0020] Compared with other drugs in clinical trials, GBP2 has the obvious advantage that it is a key molecule in the IFN-γ signaling pathway and plays an important role in clearing foreign pathogens, so patients have a high tolerance to it. GBP2 can promote the formation of an anti-tumor immune microenvironment and kill tumor cells through the IFN-γ pathway.

[0021] GBP2 can inhibit melanoma growth and improve survival rate; and promote T cells (OT-1) to kill tumors, inhibit melanoma growth and improve survival rate; GBP2 promotes the infiltration of CD8+T cells; GBP2 promotes the formation of memory T cells; GBP2 promotes the expression of immune checkpoints in tumor cells; GBP2 promotes the production of cytokines; GBP2 promotes the production of chemokine CXCL9; GBP2 promotes the increase of immunogenic molecule HMGB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 The GBP2 inhibits tumor growth in Example 1 of the present application;

[0024] Figure 2 This is the situation in Example 1 of the present application where GBP2 promotes T cells (OT-1) to inhibit tumor growth;

[0025] Figure 3 This is the situation of GBP2 extending the survival of mice in Example 2 of the present application;

[0026] Figure 4 This is the situation in Example 2 of the present application where GBP2 promotes T cells (OT-1) to prolong the survival of mice;

[0027] Figure 5 The GBP2 in Example 3 of the present application promotes the infiltration of CD8+T cells;

[0028] Figure 6 This is the situation of GBP2 promoting the formation of memory T cells in Example 4 of the present application;

[0029] Figure 7 The expression of immune checkpoints promoted by GBP2 in tumor cells in Example 5 of the present application;

[0030] Figure 8 The situation of GBP2 promoting the production of cytokine IFN-γ in Example 6 of the present application;

[0031] Fig. 9 The situation of GBP2 promoting the production of cytokine TNF-α in Example 6 of the present application;

[0032] Fig.10 This is the situation of GBP2 promoting the production of chemokine CXCL9 in Example 7 of the present application;

[0033] Fig.11 This is the situation in which GBP2 promotes the improvement of the immunogenic molecule HMGB1 in Example 7 of the present application. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present application clearer, the technical solution in the embodiments of the present application will be clearly and completely described below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application.

[0035] The embodiments of the present application provide a use of GBP2 and / or GBP2 mRNA in the preparation of a drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy.

[0036] Among them, GBP2 is guanylate binding protein 2, GBP2 mRNA can be transcribed in vivo to generate GBP2, GBP2 and / or GBP2 mRNA exist as active substances in the drug, and the drug can also contain lipid nanoparticles, which are coated on GBP2 mRNA to enhance the stability and effectiveness of drugs such as RNA vaccines.

[0037] The gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1.

[0038] The amino acid sequence of GBP2 is shown in SEQ ID NO.2.

[0039] The above-mentioned drugs can not only be used to prevent, inhibit or treat tumors, but also enhance tumor immunotherapy, such as enhancing adoptive T cell therapy.

[0040] When using the above-mentioned drug to treat tumors, a therapeutically effective amount of the above-mentioned drug containing GBP2 or lipid nanoparticles coated with GBP2 mRNA can be administered to a subject in need. Preferably, the subject is further administered tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy. Preferably, the tumor immunotherapy is administered simultaneously, sequentially or separately with the drug containing GBP2 or lipid nanoparticles coated with GBP2 mRNA.

[0041] The drug containing GBP2 or lipid nanoparticle-encapsulated GBP2 mRNA can be formulated into a dosage form for administration by oral, intradermal, subcutaneous, intraperitoneal, intravenous or intratumoral injection.

[0042] The tumor includes one or more of melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric tumors, cervical cancer, ovarian cancer, bladder cancer, urothelial carcinoma, ureteral tumors, prostate cancer, seminoma, testicular tumors, head and neck tumors, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

[0043] The embodiments of the present application also provide a drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy, wherein the drug comprises an active substance, and the active substance comprises GBP2 and / or GBP2 mRNA.

[0044] The gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1.

[0045] The amino acid sequence of GBP2 is shown in SEQ ID NO.2.

[0046] Furthermore, the drug also includes lipid nanoparticles, and the lipid nanoparticles are encapsulated outside the GBP2.

[0047] Furthermore, the tumor immunotherapy includes adoptive T cell therapy.

[0048] According to the application or medicine described in the present application, GBP2 or lipid nanoparticle-encapsulated GBP2 mRNA treats the tumor by alleviating pathological symptoms and signs, preferably by slowing down the growth rate of the tumor, and / or reducing the size of the tumor, and / or enhancing the efficacy of other treatments, and / or reducing the proportion of tumor recurrence after other treatments and / or prolonging the time of tumor recurrence after other treatments.

[0049] The term "treat" refers primarily to eliminating the disease, halting the progression of the disease, slowing the progression of the disease, reducing the duration of one or more symptoms associated with the disease, improving or reversing at least one measurable parameter associated with the disease, or increasing the survival rate of subjects suffering from the disease.

[0050] The "alleviation of pathological symptoms and signs" mentioned in this application mainly refers to the reduction of tumor masses or slowing of growth rate, relief of pain, reduction of ulcer area, reduction of bleeding, relief of anemia, relief of obstruction, and reduction of tumor infiltration and tumor metastasis.

[0051] The "slowing down of tumor growth rate" or "reducing tumor volume" mentioned in this application mainly refers to slowing down the growth rate of solid tumors that usually grow rapidly and increase significantly in volume in a short period of time, or reducing the volume of the tumor, or reducing the number of abnormal cells in hematological tumors.

[0052] The "enhancing the efficacy of other treatments" mentioned in this application mainly refers to enhancing the efficacy of other methods of treating tumors such as surgical treatment, chemotherapy, radiotherapy, immunotherapy, etc.

[0053] The "reducing the proportion of tumor recurrence after other treatments and / or prolonging the time of tumor recurrence after other treatments" mentioned in this application mainly refers to reducing the ratio of patients in whom the tumor and corresponding symptoms and signs reappear (tumor recurrence) after a period of time after the tumor is reduced or disappeared by surgical treatment, chemotherapy, radiotherapy, immunotherapy and other treatment methods; or prolonging the time interval between the reappearance of the tumor and the corresponding symptoms and signs (tumor recurrence) after the tumor is reduced or disappeared by surgical treatment, chemotherapy, radiotherapy, immunotherapy and other treatment methods.

[0054] GBP2 and / or GBP2 mRNA of the present application can be used in combination with other active ingredients, as long as they do not produce other adverse effects, such as allergic reactions, etc. GBP2 and / or GBP2 mRNA of the present application can be used as the only active ingredient or in combination with other drugs. Combination therapy is achieved by administering each treatment component simultaneously, separately or sequentially.

[0055] In other specific embodiments, GBP2 or lipid nanoparticle-encapsulated GBP2 mRNA is used in combination with one or more other treatment methods or therapeutic agents, wherein the treatment method is preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, and the therapeutic agent is preferably another agent for preventing and / or treating the occurrence and development of tumors.

[0056] The medicine of the present application can be conveniently presented in unit dosage form. The medicine of the present application can be formulated into any suitable dosage form, such as but not limited to, injection, tablet, capsule, gel, etc. The medicine of the present application can also be formulated into a suspending agent in an aqueous, non-aqueous or mixed medium. The medicine of the present application includes but is not limited to solutions, emulsions, foams and liposome-containing preparations. The medicine of the present application may include one or more penetration enhancers, carriers, excipients, and preferably the carrier is a lipid nanoparticle.

[0057] In other specific embodiments, GBP2 or lipid nanoparticle-encapsulated GBP2 mRNA is administered to a subject in a therapeutically effective amount.

[0058] The terms "patient", "subject", "individual" and the like are used interchangeably herein and refer to any human or non-human animal or its cells that can be applied to the methods described herein, preferably a human or non-human mammal. In specific embodiments, the non-human mammal includes, for example, camels, donkeys, zebras, cows, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, mice, non-human primates. In specific embodiments, the subject is a human. In specific embodiments, the subject is susceptible to, suspected of having, or has a tumor.

[0059] The term "administer" may refer to providing a predetermined substance to a subject by any appropriate method. The term "therapeutically effective amount" may refer to the amount of an active ingredient or pharmaceutical composition that induces an animal or human to show a biological or medical response considered by a researcher, veterinarian, doctor or other clinician, and this amount may include the amount of an active ingredient or pharmaceutical composition for inducing a disease or condition to be treated. It is apparent to those skilled in the art that the therapeutically effective dose and the number of administrations of the active ingredient of the present application may vary according to the desired effect. The amount or intake may be administered in a variety of dosages and methods, by distributing compositions according to the subject's weight, age, sex, health status, diet, time of administration, method of administration, excretion rate and severity of disease, such as once a day or multiple times a day.

[0060] The medicine of the present application can be applied by any general route, as long as it can reach the target tissue. It can be applied orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, endothelially, intranasally, intrapulmonaryly, rectally, intracavitarily, intraperitoneally, intrathecally and intratumorally, but is not limited thereto. In a preferred embodiment, the medicine is applied parenterally, preferably by intraperitoneal injection.

[0061] After a lot of experimental research, the applicant found that compared with other drugs in clinical trials, the obvious advantage of GBP2 is that it is a key molecule in the IFN-γ signaling pathway and plays an important role in clearing foreign pathogens, so patients have a high tolerance to it. GBP2 can promote the formation of anti-tumor immune microenvironment and kill tumor cells through the IFN-γ pathway.

[0062] This is also the case, through experiments found that:

[0063] (1) Compared with the control group, GBP2 can inhibit melanoma growth and improve survival rate; and promote T cells (OT-1) to kill tumors, inhibit melanoma growth and improve survival rate;

[0064] (2) GBP2 promoted the infiltration of CD8 + T cells compared with the control group;

[0065] (3) GBP2 promoted the formation of memory T cells compared with the control group;

[0066] (4) GBP2 promoted immune checkpoint expression in tumor cells compared with the control group;

[0067] (5) GBP2 promoted cytokine production compared with the control group;

[0068] (6) GBP2 promoted the production of chemokine CXCL9 compared with the control group;

[0069] (7) Compared with the control group, GBP2 promoted the increase of the immunogenic molecule HMGB1.

[0070] The present application is further described below through specific examples, but it should be understood that these examples are only for illustrating the present application and are not intended to limit the scope of protection of the present application in any form.

[0071] Experimental Materials:

[0072] Empty nanoparticles were lipid nanoparticles (LNPs) purchased from GenScript Biotech.

[0073] GBP2 lipid nanoparticles are GBP2 mRNA encapsulated in lipid nanoparticles and were purchased from GenScript Biotech. The preparation process is as follows:

[0074] Ionizable lipids (SM102), structural lipids (cholesterol), auxiliary lipids (DSPC) and polyethylene glycol lipids (DMG-PEG-2k) were mixed with GBP2 mRNA in acetate buffer at a volume ratio of 1:3 through a microfluidic device. The mixture was neutralized with PBS pH 7.4 buffer (containing sucrose cryoprotectant) and finally sterile filtered. After completion, the product was analyzed, and the test items included particle size, polydispersity (PDI), zeta potential, pH and endotoxin.

[0075] B16-OVA cells were donated by the Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tongji Medical College, Huazhong University of Science and Technology.

[0076] OT-1 mouse T cells were purchased from The Jackson Laboratory.

[0077] Male wild-type C57BL / 6J mice and NSG mice were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. All studies involving mice were approved by the Laboratory Animal Welfare Ethics Committee of Zhongnan Hospital of Wuhan University (ZN2024072).

[0078] Example 1: GBP2 promotes T cells (OT-1) to inhibit tumor growth.

[0079] 1. Experimental Procedure

[0080] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 4 groups (n=6 in each group).

[0081] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0082] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2);

[0083] One group of tail vein adoptive transfer 2×10 6 OT-1T cells / mL were intraperitoneally injected with empty lipid nanoparticles (OT-1+empty);

[0084] One group of tail vein adoptive transfer 2×10 6 OT-1T cells / mL were intraperitoneally injected with 40 μg / head of GBP2 lipid nanoparticles (OT-1+GBP2).

[0085] Empty nanoparticles or GBP2 lipid nanoparticles were injected every three days. Starting from the sixth day after tumor inoculation, the tumor size was measured every two days, volume = (length x width x width) / 2, and the tumor growth curve was recorded.

[0086] 2. Experimental results

[0087] For the convenience of marking and understanding, in all drawings, “empty” is represented by “Blank” and “LNP-GBP2” is represented by “GBP2”.

[0088] See also Figure 1 As shown, the tumor size of GBP2 group mice was significantly smaller than that of the empty vector group, indicating that GBP2 treatment can inhibit tumor growth; see Figure 2 As shown, the tumor size of mice in the OT-1+GBP2 group was significantly smaller than that in the OT-1+empty group, indicating that GBP2 treatment can promote T cells (OT-1) to inhibit tumor growth.

[0089] Example 2: GBP2 promotes T cells (OT-1) to kill tumors and prolongs the survival of mice.

[0090] 1. Experimental Procedure

[0091] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 4 groups (n=6 in each group).

[0092] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0093] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2);

[0094] One group of tail vein adoptive transfer 2×10 6OT-1T cells / mL were intraperitoneally injected with empty lipid nanoparticles (OT-1+empty);

[0095] One group of tail vein adoptive transfer 2×10 6 OT-1T cells / mL were intraperitoneally injected with 40 μg / head of GBP2 lipid nanoparticles (OT-1+GBP2).

[0096] Empty nanoparticles or GBP2 lipid nanoparticles were injected every three days, and the long-term survival curve of mice was recorded.

[0097] 2. Experimental results

[0098] See also Figure 3 As shown, compared with the empty group, the survival time of mice in the GBP2 treatment group was significantly prolonged, indicating that GBP2 treatment prolonged the survival time of mice; see Figure 4 As shown, compared with the OT-1+empty group, the survival of mice in the OT-1+GBP2 treatment group was significantly prolonged, indicating that GBP2 treatment promoted T cells (OT-1) to kill tumors and prolong the survival of mice.

[0099] Example 3: GBP2 promotes the infiltration of CD8+ T cells.

[0100] 1. Experimental Procedure

[0101] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 2 groups (n=6 in each group).

[0102] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0103] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2).

[0104] Empty nanoparticles or GBP2 lipid nanoparticles were injected once every three days. The long-term survival curve of mice was recorded. Mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and cut into pieces, transferred to a 50ml centrifuge tube, and 20ml of culture medium and 2mg / ml type IV collagenase were added. Digestion was performed at 37°C and 120rpm for 2-4h. After digestion, the tumor cell suspension was obtained by filtration. Immune infiltrating lymphocytes were separated using Percoll, and the proportion of CD8+T cells in the tumor was detected by flow cytometry staining.

[0105] 2. Experimental results

[0106] See also Figure 5As shown, compared with the empty group, the proportion of CD8+T cells in the tumor of mice in the GBP2 treatment group was significantly increased, indicating that GBP2 treatment promoted the tumor infiltration of CD8+T cells.

[0107] Example 4: GBP2 promotes the formation of memory T cells.

[0108] 1. Experimental Procedure

[0109] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 2 groups (n=6 in each group).

[0110] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0111] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2).

[0112] Empty nanoparticles or GBP2 lipid nanoparticles were injected every three days. Mice were killed 30 days after tumor inoculation, and lymph nodes were collected, ground, filtered, washed twice with PBS, and flow cytometry staining was used to detect the proportion of CD44+CD62L+CD8+T cells in lymph nodes.

[0113] 2. Experimental results

[0114] See also Figure 6 As shown, compared with the empty group, the proportion of CD44+CD62L+CD8+T cells in the lymph nodes of mice treated with GBP2 was significantly increased.

[0115] Example 5: GBP2 promotes immune checkpoint expression in tumor cells.

[0116] 1. Experimental Procedure

[0117] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 2 groups (n=6 in each group).

[0118] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0119] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2).

[0120] Empty nanoparticles or GBP2 lipid nanoparticles were injected every three days. Mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and 1 / 3 was separated. After fixation with 4% paraformaldehyde and embedding in paraffin, the tissue blocks were cut into a thickness of about 4 μm. The sections were deaffinity, antigen retrieval and blocking, and stained with anti-PD-L1 antibody at room temperature, and the cell nuclei were stained with DAPI. The images were recorded by fluorescence microscopy. The mean fluorescence intensity was analyzed by ImageJ software.

[0121] 2. Experimental results

[0122] See also Figure 7 As shown, the expression of immune checkpoint PD-L1 in the GBP2 treatment group was significantly increased compared with the empty group.

[0123] Example 6: GBP2 promotes the production of cytokines.

[0124] 1. Experimental Procedure

[0125] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 2 groups (n=6 in each group).

[0126] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0127] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2).

[0128] Empty nanoparticles or GBP2 lipid nanoparticles were injected once every three days. Mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and cut into pieces, transferred to a 50ml centrifuge tube, and 20ml of culture medium and 2mg / ml type IV collagenase were added. Digestion was performed at 37°C and 120rpm for 2-4h. After digestion, the tumor cell suspension was obtained by filtration. Immune infiltrating lymphocytes were separated using Percoll, and the expression of intratumoral CD8+T cell cytokines TNF-α and IFN-γ was detected by flow cytometry staining.

[0129] 2. Experimental results

[0130] See also Figure 8 and Fig. 9 As shown, compared with the empty vector group, the expression of cytokines TNF-α and IFN-γ in CD8+T cells in the tumor of mice in the GBP2 treatment group was significantly increased.

[0131] Example 7: GBP2 promotes the production of chemokine CXCL9 and immunogenic molecule HMGB1.

[0132] 1. Experimental Procedure

[0133] C57BL / 6J mice were inoculated subcutaneously on the right flank with 1 × 10 6 B16-OVA mouse melanoma cells were added per mL and the tumors were inoculated for about 5-7 days. Based on the tumor size (about 100 mm 3 ) and body weight, the mice were randomly divided into 2 groups (n=6 in each group).

[0134] One group was intraperitoneally injected with empty lipid nanoparticles (empty);

[0135] One group was intraperitoneally injected with 40 μg / mouse GBP2 lipid nanoparticles (LNP-GBP2).

[0136] Empty nanoparticles or GBP2 lipid nanoparticles were injected every three days. Mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and rinsed with pre-cooled PBS to remove residual blood (the lysed red blood cells in the homogenate will affect the measurement results). The tissue was cut into pieces after weighing. The cut tissue was placed in a corresponding volume of PBS (generally at a weight-to-volume ratio of 1:9, for example, 1g of tissue sample corresponds to 9mL of PBS, and protease inhibitors are added to PBS), and then placed in a tissue grinder and fully ground. The homogenate was centrifuged at 5000g for 10 minutes, and the supernatant was tested with ELISA kits for CXCL9 and HMGB1, respectively.

[0137] 2. Experimental results

[0138] See also Fig.10 As shown, compared with the empty group, the expression of chemokine CXCL9 in the tumor of mice in the GBP2 treatment group was significantly increased.

[0139] See also Fig.11 As shown, compared with the empty vector group, the expression of HMGB1, an immunogenic death marker, in the tumor of mice in the GBP2 treatment group was significantly increased.

[0140] SEQ ID NO.1:

[0141]

[0142]

[0143]

[0144] SEQ ID NO.2:

[0145] MASEIHMSEPMCLIENTEAQLVINQEALRILSAITQPVVVVAIVGLYRTGKSYLMNKLAGKRTGFSLGSTVQSHTKGIWMWCVPHPKKAGQTLVLLDTEGLEDVEKGDNQNDCWIFALAVLLSSTFIYNSIGTINQQAMDQLHYVTE LTDLIKSKSSPDQSGVDDSANFVGFFPTFVWTLRDFSLELEVNGKPVTSDEYLEHSLTLKKGADKKTKSFNEPRLCIRKFFPKRKCFIFDRPAQRKQLSKLETLREEELCGEFVEQVAEFTSYILSYSSVKTLCGGIIVNGPRLKSL VQTYVGAISNGSLPCMESAVLTLAQIENSAAVQKAITHYEEQMNQKIQMPTETLQELLDLHRPIESEAIEVFLKNSFKDVDQKFQTELGNLLVAKRDAFIKKNMDVSSARCSDLLEDIFGPLEEEVKLGTFSKPGGYYLFLQMRQEL EKKYNQAPGKGLQAEAMLKNYFDSKADVVETLLQTDQSLTEAAKEVEEERTKAEAAEAANRELEKKQKEFELMMQQKEKSYQEHVKKLTEKMKDEQKQLLAEQENIIAAKLREQEKFLKEGFENESKKLIREIDTLKQNKSSGKCTIL

[0146] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for herein.

Claims

1. Use of GBP2 and / or GBP2 mRNA in the preparation of a drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy.

2. The use according to claim 1, characterized in that: The gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1; And / or, the amino acid sequence of GBP2 is shown as SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The drug comprises lipid nanoparticles, and the lipid nanoparticles are encapsulated outside the GBP2 mRNA.

4. The use according to claim 1, characterized in that: The tumor immunotherapy includes adoptive T cell therapy.

5. The use according to claim 1, characterized in that: The tumor includes one or more of melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric tumors, cervical cancer, ovarian cancer, bladder cancer, urothelial carcinoma, ureteral tumors, prostate cancer, seminoma, testicular tumors, head and neck tumors, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

6. A drug for preventing, inhibiting or treating tumors or enhancing tumor immunotherapy, characterized in that: It comprises an active substance comprising GBP2 and / or GBP2 mRNA.

7. The drug according to claim 6, characterized in that: The gene sequence of the GBP2 mRNA is shown in SEQ ID NO.1; And / or, the amino acid sequence of GBP2 is shown as SEQ ID NO.

2.

8. The drug according to claim 6, characterized in that: It also includes lipid nanoparticles, wherein the lipid nanoparticles are encapsulated outside the GBP2 mRNA.

9. The drug according to claim 6, characterized in that: The tumor immunotherapy includes adoptive T cell therapy.

10. The drug according to claim 6, characterized in that: The tumor includes one or more of melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric tumors, cervical cancer, ovarian cancer, bladder cancer, urothelial carcinoma, ureteral tumors, prostate cancer, seminoma, testicular tumors, head and neck tumors, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, sarcoma, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma.

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