Use of glucose-1-phosphate in treatment of tumor

Abstract

Use of glucose-1-phosphate in the treatment of tumors. Specifically, provided are use of glucose-1-phosphate or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating tumors and use thereof in the preparation of a drug for enhancing tumor immunotherapy. In another aspect, provided is a nanoscale therapeutic agent, which comprises glucose-1-phosphate or a pharmaceutically acceptable salt thereof, a nanocarrier, and a reagent that specifically targets CD8+ T cells. The present invention relates to use of the nanoscale therapeutic agent in the preparation of a drug for treating tumors and use thereof in the preparation of a drug for enhancing tumor immunotherapy, and relates to a method for preparing the nanoscale therapeutic agent. Provided is a method for enhancing the anti-tumor activity of T cells in vitro.

Description

Use of glucose-1-phosphate in treating tumors TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine. Specifically, it relates to the use of glucose-1-phosphate in treating tumors. BACKGROUND

[0002] Tumor is a new growth of abnormal cells that multiply when a part of the body's tissue is affected by various carcinogenic factors, and the cells lose the normal regulation of their growth at the genetic level, leading to abnormal clonal proliferation. With the changes in modern social lifestyle and the improvement of people's living standards, the incidence and mortality of various malignant tumors continue to rise, posing a great threat to human health. Traditional tumor treatment methods such as surgical treatment, chemotherapy, and radiotherapy have a series of problems such as large side effects on the body, unstable treatment effect, and risk of recurrence, which cannot meet the needs of the clinic and patients.

[0003] Tumor immunotherapy is a treatment method that actively or passively stimulates or rebuilds the body's immune system to produce tumor-specific immune responses, thereby controlling and killing tumor cells. It has the advantages of high efficiency and specificity, small side effects, high safety, etc. Unlike traditional treatment methods such as surgical treatment, chemotherapy, and radiotherapy, tumor immunotherapy remodels the tumor immune microenvironment and activates the body's anti-tumor immune response to achieve the effect of eliminating tumors. In recent years, tumor immunotherapy represented by PD-1 / PD-L1 antibodies has achieved remarkable clinical efficacy in the treatment of nearly 20 kinds of solid tumors such as lung cancer, melanoma, gastrointestinal tumors, breast cancer, urinary system tumors, skin cancer, and lymphoma, 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's (allogeneic transplantation) immune cells to improve immune function. However, a part of tumor patients show treatment resistance at the initial treatment, and a part of patients who have achieved good efficacy in the early stage will still have recurrence and progression, and the number of patients who can maintain long-term benefit is very small. Therefore, overcoming the "drug resistance" of tumor immunotherapy and finding other new specific drugs are key problems that need to be solved in tumor immunotherapy. During the development of cancer, the unique metabolic mode of tumor leads to a variety of characteristics in the tumor microenvironment such as acidity, hypoxia, and lack of nutrients, which inhibits the clearance function of immune cells. Studies have shown that glycogen metabolism not only promotes the rapid response of the secondary response of memory T cells, but also is conducive to the redox homeostasis, ensuring the CD8 + The secondary response of memory T cells is an early rapid response, and it is conducive to redox homeostasis, ensuring the CD8 +High-quality response of memory T cells. In addition, studies have shown that the metabolism of glycogen and the subsequent nicotinamide adenine dinucleotide phosphate (NADPH) produced by the pentose phosphate pathway can scavenge excess reactive oxygen species (ROS) and improve the chemoresistance of tumor cells.

[0004] Glucose-1-phosphate (G1P) is a widely distributed phosphosugar in microorganisms, animal and plant cells, which participates in the biosynthesis and decomposition of glycogen in the body. In addition, G1P plays a central role in carbohydrate metabolism as an important intermediate in the interconversion of monosaccharides.

[0005] At present, there are still problems such as narrow applicability, poor prognosis, and drug resistance in the main drugs for tumor immunotherapy. Considering the key role of glycogen metabolism, immunotherapy through metabolic interference strategy can simultaneously regulate the metabolism of related immune cells in the tumor microenvironment while killing tumor cells, which is a new strategy.

[0006] SUMMARY

[0007] In one aspect, the present application provides the use of glucose-1-phosphate (G1P) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating tumors.

[0008] In another aspect, the present application provides the use of G1P or a pharmaceutically acceptable salt thereof in enhancing tumor immunotherapy. Specifically, the present application provides the use of G1P or a pharmaceutically acceptable salt thereof in the preparation of a medicament for enhancing tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy, more preferably the adoptive T cell therapy is selected from CAR-T therapy and CD8 + T cell therapy.

[0009] In another aspect, the present application provides a nanoscale therapeutic agent comprising G1P or a pharmaceutically acceptable salt thereof, a nanocarrier and a reagent specifically targeting CD8 + T cells, wherein the nanocarrier comprises polylactic acid-glycolic acid copolymer (PLGA) and maleimide polyethylene glycol polylactic acid-glycolic acid copolymer (PLGA-PEG-MAL); preferably, the reagent specifically targeting CD8 + T cells is an anti-CD8 antibody; preferably, the nanocarrier is surface-modified with streptavidin, and the reagent specifically targeting CD8 + T cells is a biotinylated anti-CD8 antibody; preferably, the nanoscale therapeutic agent has a particle size of 50-250 nm, more preferably 100-200 nm.

[0010] In another aspect, the present application provides the use of the nanoscale therapeutic agent in the preparation of a medicament for treating tumors. In another aspect, the present application provides the use of the nanoscale therapeutic agent in the preparation of a medicament for treating tumors.

[0011] In another aspect, the present application provides use of the nanoscale therapeutic agent in the manufacture of a medicament for enhancing tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy, more preferably the adoptive T cell therapy is selected from CAR-T therapy and CD8 + T cell therapy.

[0012] In another aspect, the present application provides a method of treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application, preferably the subject is further administered a tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy, more preferably the adoptive T cell therapy is selected from CAR-T therapy and CD8 + T cell therapy. Preferably, the tumor immunotherapy is administered simultaneously, sequentially or separately with G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application.

[0013] In another aspect, the present application provides a method of enhancing CD8 + T cell anti-tumor activity in vitro, the method comprising the steps of:

[0014] 1) obtaining CD8 + T cells; and

[0015] 2) contacting the CD8 + T cells with G1P or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the same.

[0016] In another aspect, the present application provides a method of preparing a nanoscale therapeutic agent, the method comprising:

[0017] 1) dissolving polylactic-co-glycolic acid (PLGA) and maleimide polyethylene glycol polylactic-co-glycolic acid (PLGA-PEG-MAL) in dichloromethane;

[0018] 2) adding glucose-1-phosphate or a pharmaceutically acceptable salt thereof to the solution of step 1);

[0019] 3) sonicating emulsification, and removing dichloromethane and large particles;

[0020] 4) optionally, adding streptavidin;

[0021] 5) optionally, adding an agent specifically targeting CD8 + T cells, the agent specifically targeting CD8 + T cells is preferably an anti-CD8 antibody, more preferably a biotinylated anti-CD8 antibody.

[0022] In some specific embodiments, the use or method according to the present application, wherein the tumor is a solid tumor, such as 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 tumor, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral tumor, prostate cancer, seminoma, testicular tumor, head and neck tumor, 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, preferably melanoma, lung cancer.

[0023] In some specific embodiments, the use or method according to the present application, wherein the G1P or a pharmaceutically acceptable salt thereof, or a nanotherapeutic agent treats the tumor by reducing pathological symptoms and signs, preferably slowing down the growth rate of the tumor, and / or reducing the volume 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; or enhancing the inhibition of tumor growth by T cells.

[0024] The term "treatment" or "treat" or "treating" or "treatments" refers primarily to eliminating the disease, preventing the disease from progressing, slowing down 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 a subject with the disease.

[0025] The "reducing pathological symptoms and signs" according to the present application refers primarily to reducing the size or slowing down the growth rate of the tumor mass, reducing pain, reducing the area of ulceration, reducing hemorrhage, reducing anemia, reducing obstruction, reducing tumor infiltration, and reducing tumor metastasis.

[0026] The "slowing down the growth rate of the tumor" or "reducing the volume of the tumor" according to the present application refers primarily to slowing down the growth rate of a solid tumor that usually grows fast and significantly increases in volume in a short period of time, or reducing the volume of the tumor. Or reducing the number of abnormal cells of a hematological tumor.

[0027] The "enhancing the efficacy of other treatments" according to the present application refers primarily to enhancing the efficacy of other methods for treating tumors, such as surgical treatment, chemotherapy, radiotherapy, immunotherapy, etc.

[0028] The "reducing the proportion of tumor recurrence after other treatment and / or prolonging the time of tumor recurrence after other treatment" described in the present application mainly refers to reducing the proportion of patients whose tumor and corresponding symptoms and signs appear again (tumor recurrence) after a period of time after the tumor is reduced or disappeared by using surgical treatment, chemotherapy, radiotherapy, immunotherapy and other treatment methods; or prolonging the time interval between the tumor and corresponding symptoms and signs appearing again (tumor recurrence) after the tumor is reduced or disappeared by using surgical treatment, chemotherapy, radiotherapy, immunotherapy and other treatment methods.

[0029] The G1P or a pharmaceutically acceptable salt thereof 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. The G1P or a pharmaceutically acceptable salt thereof of the present application can be used as the only active ingredient, or can be used in combination with other drugs. Combination therapy is achieved by administering the individual therapeutic components simultaneously, separately or sequentially.

[0030] In other specific embodiments, the G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application is used in combination with another therapeutic method or therapeutic agent, preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, and preferably another agent for preventing and / or treating tumor occurrence and development.

[0031] The G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application can be conveniently presented in unit dosage form. The G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application can be formulated into any appropriate dosage form, such as but not limited to, injections, tablets, capsules, gels, etc. The G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application can also be formulated into suspensions in aqueous, non-aqueous or mixed media. The G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application includes but is not limited to solutions, emulsions, foams and liposome-containing preparations. The G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent of the present application can include one or more penetration enhancers, carriers, excipients.

[0032] In other specific embodiments, according to the use or method described in the present application, wherein the G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent is formulated into a dosage form for administration by oral, intradermal, subcutaneous, intraperitoneal, intravenous or intratumoral injection.

[0033] In other specific embodiments, the G1P or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent is administered to a subject in a therapeutically effective amount.

[0034] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any human or non-human animal or cell thereof that can be amenable to the methods described herein, preferably a human or non-human mammal. In particular embodiments, the non-human mammal includes, for example, a camel, a donkey, a zebra, a cow, a pig, a horse, a goat, a sheep, a cat, a dog, a rat, a rabbit, a guinea pig, a mouse, a non-human primate. In particular embodiments, the subject is a human. In particular embodiments, the subject is susceptible to, suspected of having, or has a tumor.

[0035] The term "administering" can refer to providing a predetermined substance to a subject by any appropriate method. The term "therapeutically effective amount" can refer to an amount of an active ingredient or a pharmaceutical composition that induces animals or humans to exhibit a biological or medical response considered by researchers, veterinarians, doctors, or other clinicians, and such an amount can include an amount of an active ingredient or a pharmaceutical composition for inducing alleviation of a disease or a disorder to be treated. It is obvious to those skilled in the art that the therapeutically effective dose of the active ingredient of the present application and the number of administrations can vary depending on the desired effect. The administration amount or intake amount can be administered in various administration doses and methods by distributing the composition according to the body weight, age, sex, health status, diet, administration time, administration method, excretion rate, and severity of disease of the subject, for example, once a day or multiple times a day.

[0036] The G1P of the present application or a pharmaceutically acceptable salt thereof, or a nanoscale therapeutic agent can be administered by any general route as long as it can reach the target tissue. It can be administered orally, intraperitoneally, intravenously, intramuscularly, subcutaneously, endothelially, intranasally, intrapulmonarily, rectally, intracavitally, intraperitoneally, intrathecally, and intratumorally, but is not limited thereto. In a preferred embodiment, the drug is administered parenterally, preferably by intraperitoneal injection.

[0037] The present applicant prepared CD8-targeted G1P nanoparticles, confirmed their targeting property to CD8 + T cells, and found through a large number of experimental studies that,

[0038] 1) G1P treatment can inhibit tumor growth and increase survival rate compared to the control group; and promote T cell (OT-1) killing of tumors, inhibition of tumor growth, and increase of survival rate;

[0039] 2) G1P promotes the formation of memory T cells compared to the control group;

[0040] 3) G1P promotes T cell infiltration;

[0041] 4) G1P inhibits T cell exhaustion;

[0042] 5) G1P promotes cytokine production;

[0043] 6) G1P promotes T cell killing of human primary melanoma.

[0044] The obvious advantage of G1P over other drugs in clinical trials is that it is an intermediate product of glycogen metabolism itself, which is highly tolerated by patients. G1P can promote glycogen metabolism, which removes reactive oxygen species (ROS) by regulating the pentose phosphate pathway. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 shows targeting test of CD8-targeted G1P nanoparticles, administration of CD8-targeted G1P nanoparticles significantly increased the content of G1P in CD8 + T cells.

[0046] Figure 2A shows that G1P inhibits tumor growth in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0047] Figure 2B shows that G1P promotes T cells (OT-1) to inhibit tumor growth in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0048] Figure 3A shows that G1P prolongs the survival of mice in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0049] Figure 3B shows that G1P promotes T cells (OT-1) to prolong the survival of mice in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0050] Figure 4 shows that G1P promotes the formation of memory T cells in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0051] Figure 5 shows that G1P promotes T cell infiltration in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0052] Figure 6 shows that G1P inhibits T cell exhaustion in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0053] Figure 7 shows that G1P promotes cytokine production in a tumor model of mice subcutaneously inoculated with LLC-OVA lung cancer cells.

[0054] Figure 8 shows that G1P promotes CAR-T killing of tumors and inhibits tumor growth in a tumor model of mice transplanted with human primary melanoma. DETAILED DESCRIPTION

[0055] The present application is further described in the following by specific examples, but it should be understood that these examples are only intended to illustrate the present application and do not limit the scope of the present application in any form.

[0056] Examples

[0057] Experimental materials

[0058] Blank nanoparticles are CD8-targeting PLGA+PLGA-PEG-MAL blank nanoparticles: 10 mL (5 mg / mL), purchased from Shaanxi Kerry Aosheng Biotechnology Co., Ltd.

[0059] CD8-targeting G1P nanoparticles are CD8-targeting PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticles: 10 mL (5 mg / mL), purchased from Shaanxi Kerry Aosheng Biotechnology Co., Ltd.

[0060] LLC mouse Lewis lung cancer cells were purchased from the Cell Resource Center of Beijing Union Medical College.

[0061] Male wild-type C57BL / 6 mice, NSG mice, OT-1 mice were purchased from the Medical Experimental Animal Center of Chinese Academy of Medical Sciences (Beijing, China), and these animals were bred under sterile conditions in the animal facility of the Chinese Academy of Medical Sciences. All studies involving mice were approved by the Animal Care and Use Committee of the Chinese Academy of Medical Sciences (ACUC-A02-2023-091).

[0062] Human melanoma tissue samples were provided by the National Cancer Center / Cancer Hospital. Ethical permission was approved by the Medical Ethics Committee of Beijing Union Medical College (ZS2023038). All protocols complied with the Declaration of Helsinki and the Declaration of the World Medical Association, and informed consent was signed by all individuals or their family members.

[0063] Preparation Example 1. Preparation of CD8-targeting G1P nanoparticles

[0064] Materials and instruments required for the preparation of CD8-targeting G1P nanoparticles are shown in the following table:

[0065] Table 1. Preparation instruments

[0066] Table 2. Preparation materials

[0067] Preparation process:

[0068] 1. CD8-targeting PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticles (i.e., CD8-targeting G1P nanoparticles)

[0069] PLGA+PLGA-PEG-MAL was dissolved in dichloromethane, drug a-D-glucose-1- phosphate disodium salt (CAS 56401-20-8) was dissolved in deionized water and added to the dichloromethane solution, sonicated for 10 min, PVA (5%, w / v) aqueous solution was added, sonicated for 10 min, dichloromethane was removed by stirring for 1 h. Large particles were removed by centrifugation at 4000 rpm for 5 min, supernatant was removed by centrifugation at 13000 rpm for 5-10 min, the precipitate was washed with water for 2 times, then resuspended in deionized water to get PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) nanoparticle aqueous solution. PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) nanoparticle was stirred with streptavidin overnight, supernatant was removed by centrifugation at 13000 rpm for 5-10 min, the precipitate was washed with water for 2 times, then resuspended in deionized water to get PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticle aqueous solution. PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticle was stirred with biotinylated anti-CD8 antibody (ab34282) overnight, supernatant was removed by centrifugation at 13000 rpm for 5-10 min, the precipitate was washed with water for 2 times, then resuspended in deionized water to get CD8-targeted PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticle. The one that was not stirred with biotinylated anti-CD8 antibody overnight was non-targeted PLGA+PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) surface-modified streptavidin nanoparticle (i.e. non-targeted G1P nanoparticle).

[0070] 2. CD8-targeted PLGA+PLGA-PEG-MAL blank nanoparticle (i.e. blank nanoparticle)

[0071] PLGA+PLGA-PEG-MAL (25% w / w) was dissolved in dichloromethane, PVA (5%, w / v) aqueous solution was added, and ultrasonic emulsification was performed for 10 min, followed by stirring for 1 h to remove dichloromethane. Large particles were removed by centrifugation at 4000 rpm for 5 min, and the supernatant was removed by centrifugation at 13000 rpm for 5-10 min. The precipitate was washed with water twice and then resuspended in deionized water to obtain a PLGA+PLGA-PEG-MAL blank nanoparticle aqueous solution. The PLGA+PLGA-PEG-MAL blank nanoparticles were stirred overnight with streptavidin, the supernatant was removed by centrifugation at 13000 rpm for 5-10 min, the precipitate was washed with water twice, and then resuspended in deionized water to obtain a PLGA+PLGA-PEG-MAL blank nanoparticle aqueous solution modified with streptavidin on the surface. The PLGA+PLGA-PEG-MAL blank nanoparticles were stirred overnight with biotinylated anti-CD8 antibody (ab34282), the supernatant was removed by centrifugation at 13000 rpm for 5-10 min, the precipitate was washed with water twice, and then resuspended in deionized water to obtain CD8-targeted PLGA+PLGA-PEG-MAL blank nanoparticles.

[0072] Detection results:

[0073] 1. Particle size and potential of CD8-targeted G1P nanoparticles

[0074] 2. Particle size and potential of blank nanoparticles

[0075] 3. Particle size and potential of non-targeted G1P nanoparticles

[0076] Drug loading of CD8-targeted G1P nanoparticles: 7.5%; encapsulation efficiency: 50%; streptavidin modification rate: 67.5%.

[0077] Drug loading of non-targeted G1P nanoparticles: 7.8%; encapsulation efficiency: 52%.

[0078] Targeting test of CD8-targeted G1P nanoparticles: The blank nanoparticles, non-targeted G1P nanoparticles, and CD8-targeted G1P nanoparticles were added to cultured CD8 + T cells, respectively, and incubated for 12 h. The G1P content in CD8 + T cells was analyzed by liquid chromatography-mass spectrometry. It can be seen that the CD8-targeted G1P nanoparticles significantly increased the G1P content in CD8 + T cells (Figure 1).

[0079] Preparation Example 2. Preparation of OT-1 T cells

[0080] OT-1 mouse T cells were taken from OT-1 mice, using mouse CD8 + The T cell negative selection kit (purchased from Miltenyi Biotec) was used to isolate the T cells, and the procedure was as follows:

[0081] First, 3 pairs of ophthalmic scissors, 4 pairs of small forceps, 1 ml syringe or glass slide with sand, magnet and magnet holder were sterilized in the biological safety cabinet for at least 25 minutes.

[0082] After the mouse was sacrificed by cervical dislocation, it was sterilized in diluted 84 disinfectant solution for 5 minutes (tap water: 84 disinfectant solution = 4:1). The sterilized mouse was transferred to the biological safety cabinet and placed on a 10 cm culture dish with the left side facing up. The skin was lifted with forceps 1 cm below the spleen, and the mouse skin was torn upward after cutting a small opening with scissors, avoiding touching the muscle layer. New scissors and forceps were used to cut the muscle layer. A new pair of forceps was used to bluntly clamp out the entire liver, and after removing the adherent capsule and blood vessels with new scissors, the spleen was transferred to a new 10 cm culture dish.

[0083] After adding 500 ml of phosphate buffered saline (PBS) containing 2% fetal bovine serum (FBS) to the spleen, the spleen was crushed by hand with a 1 ml syringe booster until there were no obvious tissue blocks, at which time the liquid was turbid. All the liquid was transferred to a 50 ml centrifuge tube and centrifuged at 600 x g for 3 minutes at room temperature.

[0084] According to each spleen (about 1 x 10 8 The cells were resuspended by adding 400 ml of labeling buffer, and 100 μl of biotinylated antibody mixture (purchased from Miltenyi Biotec) was added, and after gentle mixing, it was incubated at 4°C in the dark for 5 minutes. After adding 300 μl of labeling buffer and 200 μl of anti-biotin microbeads, it was incubated at 4°C in the dark for 10 minutes, and during this time the magnet and magnet holder could be sterilized in the biological safety cabinet. After 8 minutes of sterilization, the LS column was placed in the middle of the magnet and placed in the appropriate position on the magnet holder, ensuring that a 15 ml centrifuge tube could be placed below it.

[0085] 1 ml of labeling buffer was added to the LS column to rinse, and the liquid flowed into the 15 ml centrifuge tube below. After all the liquid flowed out, the T cell mixture was transferred to the LS column, and after all the liquid flowed out, 500 ml of labeling buffer was added to the centrifuge tube and transferred to the LS column, then 2 ml of labeling buffer was added to the LS column, and the collected liquid flowed into the centrifuge tube. The centrifuge tube was filled with PBS containing 2% FBS, and centrifuged at 600 x g for 5 minutes at room temperature, and then washed once with PBS containing 2% FBS.

[0086] First, 25 μl / 1 x 10 6T-activator CD3 / CD28 activated magnetic beads (purchased from Thermo Fisher) and washed once with culture medium, then T cells were prepared into (0.3-1) x 10 6 cells / ml mixed solution with 20 ng / ml IL-2 and 55 μM 2-mercaptoethanol in 1640 complete culture medium. After mixing T cell mixed solution and magnetic beads, 1 ml per well was added into a 24-well plate for culture. After 48 h of activation, OT-1 mouse CD8 + T cells were obtained.

[0087] Preparation Example 3. Preparation of CAR-T cells

[0088] CAR-T cells were obtained from healthy human whole blood CD8 + T cells, which were obtained by human epidermal growth factor receptor-2 (Her2) lentivirus infection, and the specific steps were as follows:

[0089] Blood was collected in an anticoagulant tube (sodium heparin or EDTA can be used), and immediately separated or the collected healthy human peripheral blood was stored at 4°C for transportation.

[0090] RosetteSep TM human CD8 + T cell enrichment mixture (purchased from Stem cell company) antibody was directly added to the blood collection tube at 50 μl / ml blood, and after covering, it was gently inverted and mixed for 3-5 times, and was left to stand at room temperature for 20 minutes. During this period, PBS containing 2% FBS can be prepared first. Then, the separation tube was taken according to the number of blood collection tubes and the amount of blood (when the amount of blood is less than 6 ml, a 15 ml centrifuge tube is used, and when the amount of blood is more than 6 ml, it can be divided into several parts or a SepMate TM tube can be used). An appropriate amount of density gradient liquid Ficoll was added to the 15 ml centrifuge tube or SepMate TM tube.

[0091] The previously prepared PBS containing 2% FBS and blood were gently mixed at a ratio of 1:1 (or at a ratio of 2:1-3:1), and then gently blown with a pipette for 2-3 times, and the mixed solution was slowly stacked on the upper layer of the Ficoll liquid, and the Ficoll liquid was not allowed to be dispersed by the mixed solution. The lid was covered, and it was carefully transferred to the centrifuge, and centrifuged at 1200g at room temperature for 20 minutes.

[0092] After centrifugation, carefully transfer to the biosafety cabinet, and use a gun or a Pasteur pipette to suck the middle white film layer and transfer it to a new centrifuge tube. Add 1% FBS PBS to the centrifuge tube and wash once at 600g for 10 minutes at room temperature. If there are red blood cells, break them and wash again, or if not, wash again at 500g for 5 minutes at room temperature. Count after adding X-VIVO15 complete medium containing 20ng / ml (100U / ml) interleukin 2 (IL-2), and then add 20ng / ml interleukin 7 (IL-7), and freeze at 1x10 6

[0093] When needed immediately, first take 25μl / 1x10 6 6 The T cell mixture is mixed with the magnetic beads and added to a 24-well plate at 1ml per well.

[0094] Prepare the coating solution according to the ratio of 10μl 1μg / ml Retronectin in 250μl PBS, and add it to the 24-well plate and incubate at room temperature for at least 30 minutes. Discard the coating solution before use and rinse with PBS once. After 24 hours of activation, transfer the T cells to the Retronectin-coated 24-well plate and continue to culture for 24 hours.

[0095] Slowly add the corresponding CAR lentivirus directly to the T cell culture solution, gently change the liquid after 6-8 hours of infection, and do not blow the adherent T cells. Continue to culture for 24 hours and then transfer to a culture bottle.

[0096] The preparation process of CAR lentivirus is as follows:

[0097] PsPAX2 (Plasmid 12260), pMD2.G (Plasmid 12259) plasmids are purchased from Addgene (MA, USA), and Her2-specific CAR T plasmid (Professor Zhang Yi of the First Affiliated Hospital of Zhengzhou University) is laboratory-owned.

[0098] Prepare the coating solution under sterile conditions according to the ratio of polylysine:gelatin = 1:4, add 5ml of coating solution per bottle, and then add overnight, rinse with PBS once before use (or incubate at room temperature for 1 hour, then recover the coating solution, replace it with PBS, and discard the PBS directly before use).

[0099] ​​Thaw the 293T cell line in a 37°C water bath, and shake repeatedly to accelerate the thawing process. After the ice in the cryopreservation tube completely melts, transfer the cells to a 15-ml centrifuge tube containing 5 ml of DMEM complete medium, and centrifuge at 1500 rpm for 5 min at room temperature. Discard the supernatant, resuspend the cells, and transfer them to a pre-coated T25 culture bottle. Add DMEM complete medium to a total volume of 10 ml. When the cells grow to 90-95% confluence, subculture them according to a one-to-three ratio.

[0100] Calculate the amount of plasmid and prepare the mixture. Prepare the lentivirus (LV) according to the desired plasmid Her2-specific CART plasmid (50%): envelope plasmid pSpAX2 (35%): packaging plasmid pMD2.G (15%) = 10:7:3. That is, add 10 μg of the desired plasmid, 7 μg of pSpAX2 plasmid, and 3 μg of pMD2.G plasmid to a T25 culture bottle. According to the amount of 250 μl of Opti MEM medium added to each T25 bottle, add an appropriate amount of Opti MEM medium to a 15-ml centrifuge tube, and then add the plasmids as needed. Let it stand for 5 minutes.

[0101] Prepare the transfection reagent mixture: add Opti MEM at 250 μl / bottle, then add the transfection reagent Viafect (according to the plasmid total amount ng: transfection reagent μl = 1:3) to a 15-ml centrifuge tube, mix well, and let it stand for 5 minutes.

[0102] After standing, transfer the transfection reagent mixture to the plasmid mixture at 250 μl / bottle, mix well, and let it stand for 15 minutes. Gently aspirate the mixture, and add it dropwise to the cells at 500 μl / bottle, mix gently, and label the date and the name of the desired plasmid.

[0103] After 6-8 hours of transfection, gently replace the DMEM medium, and add sodium butyrate (final concentration 1 mM) prepared on site. Collect the virus once at 48 hours and 72 hours after medium replacement, concentrate the virus using a 100-kDa concentrator tube, label it, and transfer it to -80°C for storage.

[0104] Example 1. G1P promotes T cells (OT-1) to inhibit tumor growth.

[0105] 1. Experimental procedure

[0106] C57BL / 6J mice were subcutaneously inoculated with 1×10 6 LLC-OVA lung cancer cells on the ventral side, and eight days after tumor inoculation, the mice were randomly divided into four groups (n = 6 per group) based on tumor size and body weight. One group was injected intraperitoneally with blank nanoparticles (blank); one group was injected intraperitoneally with 50 μg / kg of CD8-targeted G1P nanoparticles (G1P); one group was injected intravenously with 2×10 6OT-1 T cells, and intraperitoneally injected with blank nanoparticles (OT1+blank); one group of mice was injected with 2x10 6 OT-1 T cells, and intraperitoneally injected with 50 μg / kg of CD8-targeted G1P nanoparticles (OT1+G1P). The blank nanoparticles or CD8-targeted G1P nanoparticles were injected once every two days. After the eighth day of tumor inoculation, the tumor size was measured once every two days, and the tumor growth curve was recorded.

[0107] 2. Experimental results

[0108] The tumor size of the G1P group of mice was significantly smaller than that of the blank group (Figure 2A), indicating that G1P treatment can inhibit the growth of tumors; the tumor size of the OT1+G1P group of mice was significantly smaller than that of the OT1+blank group (Figure 2B), indicating that G1P treatment can promote T cells (OT-1) to inhibit the growth of tumors.

[0109] Example 2. G1P promotes T cells (OT-1) to kill tumors and prolong the survival of mice.

[0110] 1. Experimental steps

[0111] C57BL / 6J mice were inoculated with 1x10 6 LLC-OVA lung cancer cells on the ventral side, and after eight days of tumor inoculation, the mice were randomly divided into four groups (n=6 for each group) based on tumor size and body weight. One group of mice was intraperitoneally injected with blank nanoparticles (blank); one group of mice was intraperitoneally injected with 50 μg / kg of CD8-targeted G1P nanoparticles (G1P); one group of mice was injected with 2x10 6 OT-1 T cells, and intraperitoneally injected with blank nanoparticles (OT1+blank); one group of mice was injected with 2x10 6 OT-1 T cells, and intraperitoneally injected with 50 μg / kg of CD8-targeted G1P nanoparticles (OT1+G1P). The blank nanoparticles or CD8-targeted G1P nanoparticles were injected once every two days. The long-term survival curve of the mice was recorded.

[0112] 2. Experimental results

[0113] Compared with the blank group, the survival of the mice in the G1P treatment group was significantly prolonged (Figure 3A), indicating that G1P treatment prolongs the survival of mice; compared with the OT1+blank group, the survival of the mice in the OT1+G1P treatment group was significantly prolonged (Figure 3B), indicating that G1P treatment promotes T cells (OT-1) to prolong the survival of mice.

[0114] Example 3. G1P promotes the formation of memory T cells.

[0115] 1. Experimental steps

[0116] C57BL / 6J mice were inoculated subcutaneously on the ventral side with 1×10 6 Eight days after tumor inoculation with LLC-OVA mice with lung cancer cells, mice were randomly divided into two groups (n=6 per group) based on tumor size and body weight. One group received intraperitoneal injections of blank nanoparticles (Blank); the other group received intraperitoneal injections of 50 μg / kg of CD8-targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected every two days. Thirty days after tumor inoculation, mice were sacrificed, and lymph nodes were harvested, ground, filtered, and washed twice with PBS.

[0117] CD44 plays an important role in the formation and maintenance of long-term immune memory, helping T cells to quickly initiate a response when they encounter the same antigen again. CD62L is considered a marker of central memory T cells. These cells have a high expression of CD62L and are able to quickly locate to locations such as lymph nodes in order to quickly respond again. Flow cytometric staining to detect CD44 in lymph nodes + CD62L + The proportion of T cells.

[0118] 2. Experimental Results

[0119] 30 days after tumor inoculation, i.e. 22 days after the injection of blank nanoparticles or CD8-targeted G1P nanoparticles, the number of CD44 in the lymph nodes of mice in the G1P-treated group was significantly higher than that in the blank group. + CD62L + The proportion of T cells increased significantly (Figure 4).

[0120] Example 4. G1P promotes T cell infiltration.

[0121] 1. Experimental Procedure

[0122] C57BL / 6J mice were inoculated subcutaneously on the ventral side with 1×10 6 LLC-OVA mouse lung cancer cells were inoculated with the tumor. Eight days after the tumor was inoculated, the mice were randomly divided into two groups (n=6 in each group) based on tumor size and body weight. One group was intraperitoneally injected with blank nanoparticles (blank); the other group was intraperitoneally injected with 50μg / kg of CD8-targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected once every two days. Mice were killed 30 days after tumor inoculation, and the subcutaneous tumor tissue was peeled off and minced, transferred to a 50ml centrifuge tube, and 20ml of culture medium and 2mg / ml type IV collagenase were added. Digestion was carried out 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 CD8 in the tumor was detected by flow cytometry. + The proportion of T cells.

[0123] 2. Experimental Results

[0124] Intratumoral CD8 T cells were significantly increased in G1P treated mice compared with blank treated mice at the time of tumor inoculation (30 days) and the start of injection of blank nanoparticles or CD8 targeted G1P nanoparticles (22 days) (Fig. 4). + The proportion of T cells was significantly increased (Fig. 5), indicating that G1P treatment promoted tumor infiltration of T cells.

[0125] Example 5. G1P inhibits the exhaustion of T cells.

[0126] 1. Experimental steps

[0127] C57BL / 6J mice were subcutaneously inoculated with 1 x 10 6 After eight days of tumor inoculation, the mice were randomly divided into two groups (n = 6 in each group) based on tumor size and body weight. One group was injected intraperitoneally with blank nanoparticles (blank), and one group was injected intraperitoneally with 50 μg / kg of CD8 targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8 targeted G1P nanoparticles were injected every two days. After 30 days of tumor inoculation, the mice were sacrificed, and the subcutaneous tumor tissue was peeled off and cut into small pieces, transferred to a 50 ml centrifuge tube, and 20 ml of medium and 2 mg / ml of collagenase type IV were added. The mixture was digested at 37°C and 120 rpm for 2-4 h. After digestion, the tumor cell suspension was obtained by filtration. Immune infiltrating lymphocytes were obtained by Percoll separation.

[0128] PD1, TIM3, and LAG3 are markers of T cell exhaustion and are highly expressed in exhausted T cells. Flow cytometry staining was used to detect intratumoral CD8 + The expression of T cell immune checkpoints PD1, TIM3, and LAG3.

[0129] 2. Experimental results

[0130] Intratumoral CD8 T cells were significantly increased in G1P treated mice compared with blank treated mice at the time of tumor inoculation (30 days) and the start of injection of blank nanoparticles or CD8 targeted G1P nanoparticles (22 days) (Fig. 4). + The expression of T cell immune checkpoints PD1, TIM3, and LAG3 was significantly decreased (Fig. 6).

[0131] Example 6. G1P promotes the production of cytokines.

[0132] 1. Experimental steps

[0133] C57BL / 6J mice were subcutaneously inoculated with 1 x 10 6LLC-OVA lung cancer cells, after eight days of tumor inoculation, mice were randomly divided into two groups (n=6 per group) based on tumor size and body weight. One group was injected intraperitoneally with blank nanoparticles (blank); one group was injected intraperitoneally with 50 μg / kg of CD8-targeted G1P nanoparticles (G1P). The blank nanoparticles or CD8-targeted G1P nanoparticles were injected once every two days. After 30 days of tumor inoculation, the mice were sacrificed, and the subcutaneous tumor tissues were peeled off and cut into small pieces, transferred to a 50 ml centrifuge tube, 20 ml of medium and 2 mg / ml collagenase type IV were added, and digested at 37°C, 120 rpm for 2-4 h. After digestion, the tumor cell suspension was obtained by filtration. Immune infiltrating lymphocytes were obtained by Percoll separation, and the intratumoral CD8 + T cell cytokines TNF-a and IFN-g expression.

[0134] 2. Experimental results

[0135] At 30 days of tumor inoculation, i.e., 22 days of injection of blank nanoparticles or CD8-targeted G1P nanoparticles, compared with the blank group, the intratumoral CD8 + T cell cytokines TNF-a and IFN-g expression increased significantly (Figure 7).

[0136] Example 7. G1P promotes CAR-T killing of tumors and inhibits tumor growth.

[0137] 1. Experimental steps

[0138] Human melanoma tissues (F0 generation) were washed three times in PBS containing penicillin / streptomycin, and the entire specimen was cut into tissues of approximately 5 x 5 mm in size. Individual tumor tissues were implanted into the right flank of anesthetized NSG mice to generate F1 generation. After the F1 generation tumor was implanted, when the tumor grew to 1 cm 3 in size, it was excised and cut into multiple tissues (3 x 3 mm) and transplanted into new NSG mice to generate the next generation. After 3 generations, the mice were randomly divided into different groups, and CAR-T cells (1 x 10 6 cells per mouse) were injected into the tail vein after the F4 generation tumor grew to 5 x 5 mm.

[0139] The mice were randomly divided into two groups (n=6 per group). One group was injected intraperitoneally with blank nanoparticles (blank); one group was injected intraperitoneally with 50 μg / kg of CD8-targeted G1P nanoparticles (G1P). The blank nanoparticles or CD8-targeted G1P nanoparticles were injected once every two days, and the tumor growth curve was recorded.

[0140] 2. Experimental results

[0141] The tumor size of the G1P treatment group was significantly smaller than that of the blank group (Figure 8), indicating that G1P treatment promotes CAR-T killing of tumors and inhibits tumor growth.

[0142] The results of the above examples show that:

[0143] CD8-targeted G1P nanoparticles were prepared, and their targeting of CD8 + T cells was confirmed.

[0144] G1P can effectively enhance T cell function and is a new strategy for tumor immunotherapy.

[0145] In vivo studies found that, compared with the control group, G1P treatment can inhibit tumor growth and improve survival rate; and promote T cells (OT-1) to kill tumors, inhibit tumor growth and improve survival rate; compared with the control group, G1P promotes the formation of memory T cells; G1P promotes T cell infiltration; G1P inhibits T cell exhaustion; G1P promotes cytokine production; G1P promotes CAR-T killing of tumors and inhibits tumor growth.

[0146] Compared with other drugs in clinical trials, the obvious advantage of G1P is that it is an intermediate product of glycogen metabolism, and patients have high tolerance to it. G1P can promote glycogen metabolism, which removes reactive oxygen species (ROS) by regulating the phosphopentose pathway.

Claims

1. Use of glucose-1 -phosphate or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of a tumor.

2. Use of glucose-1-phosphate or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for enhancing tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy, more preferably the adoptive T cell therapy is selected from the group consisting of CAR-T therapy and CD8 + T cell therapy.

3. A nanoscale therapeutic agent comprising glucose- 1 -phosphate or a pharmaceutically acceptable salt thereof, a nanocarrier, and an agent that specifically targets CD8 + T cells, the nanocarrier comprising poly(lactic-co-glycolic acid) (PLGA) and maleimide polyethylene glycol poly(lactic-co-glycolic acid) (PLGA-PEG-MAL); Preferably, the specific targeting of CD8 + The agent for T cells is an anti-CD8 antibody; Preferably, the nanocarriers are surface-modified with streptavidin, and the specific targeting CD8 + The reagent for T cells is a biotinylated anti-CD8 antibody; Preferably, the nanoscale therapeutic agent has a particle size of 50-250 nm, more preferably 100-200 nm.

4. Use of a nanoscale therapeutic agent according to claim 3 for the manufacture of a medicament for the treatment of a tumor.

5. Use of a nanoscale therapeutic agent according to claim 3 for the manufacture of a medicament for enhancing tumor immunotherapy, preferably the tumor immunotherapy is adoptive T cell therapy, more preferably the adoptive T cell therapy is selected from the group consisting of CAR-T therapy and CD8 + T cell therapy.

6. Use according to any one of claims 1 to 2 and 4 to 5, wherein the tumor is a solid tumor, such as melanoma, lung cancer, skin cancer, liver cancer, kidney cancer, nasopharyngeal cancer, gastric cancer, esophageal cancer, colorectal cancer, colon cancer, rectal cancer, gallbladder cancer, cholangiocarcinoma, choriocarcinoma, pancreatic cancer, pediatric tumor, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral tumor, prostate cancer, seminoma, testicular tumor, head and neck tumor, 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, preferably melanoma, lung cancer.

7. Use according to any one of claims 1 to 2 and 4 to 5, wherein the medicament is used in combination with another therapeutic method or therapeutic agent, preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, preferably another agent for the treatment of a tumor; preferably the therapeutic method or therapeutic agent is administered simultaneously, sequentially or separately from the glucose-1 -phosphate or the pharmaceutical composition containing the same.

8. Use according to any one of claims 1 to 2 and 4 to 5, wherein the medicament treats the tumor by alleviating pathological symptoms and signs, preferably slowing down the growth rate of the tumor, and / or reducing the volume 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; or enhancing the inhibitory effect of T cells on tumor growth.

9. A method of preparing the nanoscale therapeutic agent of claim 3, the method comprising: 1) dissolving polylactic-co-glycolic acid (PLGA) and maleimide polyethylene glycol polylactic-co-glycolic acid (PLGA-PEG-MAL) in dichloromethane, 2) adding glucose-1 -phosphate or a pharmaceutically acceptable salt thereof to the solution of step 1), and 3) sonicating and removing dichloromethane and large particles; 4) optionally, adding streptavidin; 5) optionally, adding a reagent that specifically targets CD8 + T cells, said reagent that specifically targets CD8 + The reagent that specifically targets CD8 T cells is preferably an anti-CD8 antibody, more preferably a biotinylated anti-CD8 antibody.

10. A method of enhancing T cell anti-tumor activity in vitro, the method comprising the steps of: 1) obtaining T cells; and 2) contacting the T cells with glucose-1 -phosphate or a pharmaceutically acceptable salt thereof, a pharmaceutical composition containing the same or the nanoscale therapeutic agent of claim 3, wherein said T cells are preferably CD8 + T cells.

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