WO2025217985A1 - Use of glucose-1-phosphate in treatment of tumor - Google Patents

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

The present application relates to the field of biomedicine, and more particularly to the use of glucose-1-phosphate in treating tumors.

Background Art

Tumors are neoplasms formed when, under the influence of various carcinogenic factors, a single cell in a localized tissue loses its normal genetic regulation of growth, leading to clonal abnormal proliferation. With changes in modern lifestyles and improvements in living standards, the morbidity and mortality rates of various malignant tumors continue to rise, posing a significant threat to human health. Traditional tumor treatments, such as surgery, chemotherapy, and radiotherapy, are no longer able to meet clinical and patient needs due to significant side effects, unstable therapeutic effects, and the risk of recurrence.

Tumor immunotherapy refers to a treatment approach that actively or passively stimulates or reprograms the body's immune system to generate a tumor-specific immune response, thereby controlling and killing tumor cells. It boasts the advantages of high efficacy, specificity, minimal side effects, and a high safety profile. Unlike traditional treatments such as surgery, chemotherapy, and radiotherapy, tumor immunotherapy reshapes the tumor's immune microenvironment and activates the body's anti-tumor immune response, thereby achieving tumor eradication. In recent years, tumor immunotherapy, exemplified by PD-1/PD-L1 antibodies, has achieved remarkable clinical efficacy in the treatment of nearly 20 solid tumors, including lung cancer, melanoma, gastrointestinal cancers, breast cancer, urinary tract cancers, skin cancer, and lymphoma, marking a milestone in cancer treatment. Adoptive T cell therapy is an immunotherapy that harnesses a patient's own immune cells to detect and eliminate tumor cells. It utilizes either the patient's own immune cells (autologous transplantation) or a donor's (allogeneic transplantation) to enhance immune function. However, some cancer patients exhibit resistance to initial treatment, and even those who achieve good initial responses will experience relapse and progression. Only a handful of patients achieve sustained long-term benefits. Therefore, overcoming the "drug resistance" of tumor immunotherapy and finding other new and effective drugs are key issues that need to be addressed in tumor immunotherapy. During the development of cancer, the unique metabolic mode of the tumor leads to multiple characteristics in the tumor microenvironment, such as acidity, hypoxia, and nutrient deficiency, which inhibit the clearance function of immune cells. Studies have shown that glycogen metabolism not only promotes The secondary response of CD8 ⁺ memory T cells is rapid and early, and it also promotes redox homeostasis, ensuring a high-quality response of CD8 ⁺ memory T cells. Furthermore, studies have shown that glycogen metabolism and the subsequent production of nicotinamide adenine dinucleotide phosphate (NADPH) by the pentose phosphate pathway can scavenge excess reactive oxygen species (ROS) and improve chemotherapy resistance in tumor cells.

Glucose-1-phosphate (G1P) is a sugar phosphate widely distributed within microbial, animal, and plant cells. It participates in the biosynthesis and degradation of glycogen within organisms. Furthermore, G1P plays a central role in carbohydrate metabolism, serving as an important intermediate in the interconversion of monosaccharides.

Currently, mainstream cancer immunotherapy drugs still face challenges in clinical practice, such as limited applicability, poor prognosis, and drug resistance. Considering the critical role of glycogen metabolism, immunotherapy using metabolic interference strategies is a novel approach that can simultaneously regulate the metabolism of related immune cells in the tumor microenvironment while simultaneously killing tumor cells.

Summary of the Invention

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

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.

On the other hand, the present application provides a nanoscale therapeutic agent, which comprises G1P or a pharmaceutically acceptable salt thereof, a nanocarrier and an agent that specifically targets 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 agent that specifically targets CD8 ⁺ T cells is an anti-CD8 antibody; preferably, the surface of the nanocarrier is modified with streptavidin, and the agent that specifically targets CD8 ⁺ T cells is a biotinylated anti-CD8 antibody; preferably, the particle size of the nanoscale therapeutic agent is 50-250 nm, more preferably 100-200 nm.

On the other hand, the present application provides the nanoscale therapeutic agent in the preparation of a method for treating tumors. Use in medicine for tumors.

On the other hand, the present application provides the use of the nanoscale therapeutic agent in the preparation of a drug 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.

In another aspect, the present application provides a method for treating tumors, comprising administering a therapeutically effective amount of G1P or a pharmaceutically acceptable salt thereof, or the nanoscale therapeutic agent of the present application to a subject in need thereof, preferably further administering tumor immunotherapy to the subject, 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 the nanoscale therapeutic agent of the present application.

In another aspect, the present application provides a method for enhancing the anti-tumor activity of CD8 ⁺ T cells in vitro, the method comprising the following steps:

1) obtaining CD8 ⁺ T cells; and

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

In another aspect, the present application provides a method for preparing a nanoscale therapeutic agent, the method comprising:

1) dissolving poly(lactic acid-co-glycolic acid) (PLGA) and poly(maleimide-polyethylene glycol) poly(lactic acid-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);

3) ultrasonic emulsification and removal of dichloromethane and large particles;

4) Optionally, add streptavidin;

5) Optionally, a reagent that specifically targets CD8 ⁺ T cells is added. The reagent that specifically targets CD8 ⁺ T cells is preferably an anti-CD8 antibody, more preferably a biotinylated anti-CD8 antibody.

In some specific embodiments, according to the uses or methods described herein, 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 cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral cancer, prostate cancer, seminoma, testicular tumor, head and neck tumor, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, Sarcoma, bone tumor, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma, preferably melanoma, lung cancer.

In other specific embodiments, according to the uses or methods described herein, the G1P or a pharmaceutically acceptable salt thereof, or the nanotherapeutic agent treats the tumor by: alleviating pathological symptoms and signs, preferably slowing 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; or enhancing the inhibitory effect of T cells on tumor growth.

The term "treat" generally refers to eliminating the disease, arresting 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 of subjects suffering from the disease.

The "relief 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.

The term "slowing down tumor growth" or "reducing tumor volume" as used herein refers primarily to slowing down the growth of solid tumors that typically grow rapidly and increase significantly in volume within a short period of time, or reducing the volume of the tumor. Alternatively, it may refer to reducing the number of abnormal cells in hematologic tumors.

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.

The "reduction in the proportion of tumor recurrence after other treatments and/or prolongation of the time to 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 means; 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 means.

The G1P or its pharmaceutically acceptable salts 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. The G1P or its pharmaceutically acceptable salts of the present application can be used as the sole active ingredient or in combination with other drugs. Combination therapy is achieved by administering the individual therapeutic components simultaneously, separately, or sequentially.

In other specific embodiments, the G1P or its pharmaceutically acceptable salt, or nanoscale therapeutic agent of the present application is used in combination with one or more other treatment methods or therapeutic agents, wherein the treatment method is preferably radiotherapy, chemotherapy, immunotherapy, or targeted therapy, and the therapeutic agent is preferably another agent for preventing and/or treating the occurrence and development of tumors.

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

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

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

The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any human or non-human animal, or cells thereof, suitable for use with 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, and non-human primates. In specific embodiments, the subject is a human. In specific embodiments, the subject is susceptible to, suspected of having, or already has a tumor.

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 exhibit a biological or medical response considered by a researcher, veterinarian, doctor or other clinician, and such an amount may include the amount of an active ingredient or pharmaceutical composition used to induce relief of the disease or condition to be treated. It is obvious to those skilled in the art that the therapeutically effective dose and number of administrations of the active ingredients of the present application may vary depending on the desired effect. The administration amount or intake amount may be administered in a variety of dosages and methods, by adjusting the dosage according to the subject's weight, age, sex, health status, diet, administration time, administration method, excretion, etc. The composition may be dispensed according to the rate of excretion and severity of the disease, for example, once a day or multiple times a day.

The G1P or pharmaceutically acceptable salt thereof, or nanoscale therapeutic agent of the present application can be administered by any common route, as long as it can reach the target tissue. Administration can be oral, intraperitoneal, intravenous, intramuscular, subcutaneous, endothelial, intranasal, intrapulmonary, rectal, intracavitary, intraperitoneal, intrathecal, and intratumoral, but is not limited thereto. In a preferred embodiment, the drug is administered parenterally, preferably by intraperitoneal injection.

The applicant prepared CD8-targeted G1P nanoparticles, confirmed their targeting to CD8 ⁺ T cells, and found through extensive experimental studies that

1) Compared with the control group, G1P treatment can inhibit tumor growth and improve survival rate; it also promotes T cells (OT-1) to kill tumors, inhibit tumor growth and improve survival rate;

2) G1P promoted the formation of memory T cells compared with the control group;

3) G1P promotes T cell infiltration;

4) G1P inhibits T cell exhaustion;

5) G1P promotes cytokine production;

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

Compared to other drugs in clinical trials, G1P has a significant advantage in that it is an intermediate product of glycogen metabolism and is well tolerated by patients. G1P may promote glycogen metabolism, which in turn scavenges reactive oxygen species (ROS) by regulating the pentose phosphate pathway.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows the targeting test of CD8-targeted G1P nanoparticles. Administration of CD8-targeted G1P nanoparticles significantly increased the G1P content in CD8 ⁺ T cells.

FIG2A shows that G1P inhibits tumor growth in a tumor model in which LLC-OVA lung cancer cells were subcutaneously inoculated in mice.

FIG2B shows that G1P promotes T cells (OT-1) to inhibit tumor growth in a tumor model in which mice were subcutaneously inoculated with LLC-OVA lung cancer cells.

FIG3A shows that G1P prolongs the survival of mice in a tumor model in which LLC-OVA lung cancer cells were subcutaneously inoculated in mice.

FIG3B shows that G1P promotes T cells (OT-1) to prolong the survival of mice in a tumor model in which LLC-OVA lung cancer cells were subcutaneously inoculated.

Figure 4 shows that G1P plays an important role in the tumor model of LLC-OVA lung cancer cells in mice subcutaneously inoculated Promotes the formation of memory T cells.

FIG5 shows that G1P promotes T cell infiltration in a tumor model in which LLC-OVA lung cancer cells were subcutaneously inoculated in mice.

FIG6 shows that G1P inhibits T cell exhaustion in a tumor model in which mice were subcutaneously inoculated with LLC-OVA lung cancer cells.

FIG7 shows that G1P promotes cytokine production in a tumor model in which LLC-OVA lung cancer cells were subcutaneously inoculated in mice.

Figure 8 shows that G1P promotes CAR-T cell killing of tumors and inhibits tumor growth in a mouse model of human primary melanoma transplanted with human melanoma.

DETAILED DESCRIPTION

The present invention is further described below by means of specific examples. However, it should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention in any form.

Example

Experimental Materials

The blank nanoparticles were CD8-targeted PLGA+PLGA-PEG-MAL blank nanoparticles: 10 mL (5 mg/mL), purchased from Shaanxi Kairui Aosheng Biotechnology Co., Ltd.

CD8-targeted G1P nanoparticles are CD8-targeted PLGA+PLGA-PEG-MAL drug-loaded (CAS56401-20-8) surface-modified streptavidin nanoparticles: 10 mL (5 mg/mL), purchased from Shaanxi Kairui Aosheng Biotechnology Co., Ltd.

LLC mouse Lewis lung cancer cells were purchased from the Cell Resource Center of Peking Union Medical College.

Male wild-type C57BL/6 mice, NSG mice, and OT-1 mice were purchased from the Medical Laboratory Animal Center of the Chinese Academy of Medical Sciences (Beijing, China). These animals were maintained 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).

Human melanoma tissue samples were provided by the National Cancer Center/Cancer Hospital. Ethical approval was obtained from the Medical Ethics Committee of Peking Union Medical College (ZS2023038). All protocols adhered to the Declaration of Helsinki and the Declaration of the World Medical Association, and informed consent was obtained from all individuals or their families.

Preparation Example 1. Preparation of CD8-targeted G1P nanoparticles

The materials and instruments required for the preparation of CD8-targeted G1P nanoparticles are shown in the table below:

Table 1. Preparation instruments

Table 2. Preparation materials

Preparation process:

1. CD8-targeted PLGA+PLGA-PEG-MAL drug-loaded (CAS56401-20-8) surface-modified streptavidin nanoparticles (i.e., CD8-targeted G1P nanoparticles)

PLGA + PLGA-PEG-MAL was dissolved in dichloromethane. The drug α-D-glucose-1-phosphate disodium salt (CAS 56401-20-8) was dissolved in deionized water and added to the dichloromethane solution. Ultrasonic emulsification was performed for 10 minutes. A 5% w/v aqueous solution of PVA was added and ultrasonic emulsification was performed for 10 minutes. The dichloromethane was removed by stirring for 1 hour. The mixture was centrifuged at 4000 rpm for 5 minutes to remove large particles. The supernatant was removed by centrifugation at 13000 rpm for 5-10 minutes, and the precipitate was washed twice with water and resuspended in deionized water to obtain an aqueous solution of PLGA + PLGA-PEG-MAL drug-loaded (CAS 56401-20-8) nanoparticles. The PLGA+PLGA-PEG-MAL drug-loaded (CAS56401-20-8) nanoparticles were stirred with streptavidin overnight, centrifuged at 13000 rpm for 5-10 min, the supernatant was removed, the precipitate was washed twice with water, and then resuspended in deionized water to obtain an aqueous solution of PLGA+PLGA-PEG-MAL drug-loaded (CAS56401-20-8) surface-modified streptavidin nanoparticles. The nanoparticles were stirred overnight with a biotinylated anti-CD8 antibody (ab34282), centrifuged at 13,000 rpm for 5-10 minutes, and the supernatant removed. The precipitate was washed twice with water and then resuspended in deionized water to obtain CD8-targeted PLGA + PLGA-PEG-MAL drug-loaded (CAS56401-20-8) surface-modified streptavidin nanoparticles. Those not stirred overnight with the biotinylated anti-CD8 antibody were non-targeted PLGA + PLGA-PEG-MAL drug-loaded (CAS56401-20-8) surface-modified streptavidin nanoparticles (i.e., non-targeted G1P nanoparticles).

2. CD8-targeted PLGA+PLGA-PEG-MAL blank nanoparticles (i.e., blank nanoparticles)

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

Test results:

1. Particle Size and Potential of CD8-Targeted G1P Nanoparticles

2. Particle size and potential of blank nanoparticles

3. Particle Size and Potential of Non-targeted G1P Nanoparticles

The drug loading capacity of CD8-targeted G1P nanoparticles was 7.5%, the encapsulation efficiency was 50%, and the streptavidin modification rate was 67.5%.

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

Targeting test of CD8-targeted G1P nanoparticles: Blank nanoparticles, non-targeted G1P nanoparticles, and CD8-targeted G1P nanoparticles were added to cultured CD8 ⁺ T cells. After incubation for 12 hours, the G1P content in the CD8 ⁺ T cells was analyzed by liquid chromatography-mass spectrometry. It can be seen that the administration of CD8-targeted G1P nanoparticles significantly increased the G1P content in CD8 ⁺ T cells (Figure 1).

Preparation Example 2. Preparation of OT-1T cells

OT-1 mouse T cells were isolated from OT-1 mice using a mouse CD8 ⁺ T cell negative isolation kit (purchased from Miltenyi Biotec). The specific steps are as follows:

First, place three ophthalmic scissors, four small tweezers, a 1ml syringe or a sand-filled slide, a magnet stand, and a 10cm culture dish in a biosafety cabinet and sterilize for at least 25 minutes.

After killing the mouse by cervical dislocation, place it in diluted 84 disinfectant for disinfection for 5 minutes (tap water: 84 disinfectant = 4:1). Transfer the disinfected mouse to the biosafety cabinet and place it in a 10cm culture dish with its left side facing up. Use tweezers to lift the skin 1cm below the spleen, cut a small incision with scissors, and tear the mouse skin upwards, making sure not to touch the muscle layer. Use new scissors and tweezers to cut the muscle layer in the same way. Use new tweezers to bluntly clamp out the entire liver, and use new scissors to remove the adherent bag. After removing the membranes and blood vessels, transfer the spleen to a new 10 cm culture dish.

After adding 500 ml of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS) to the spleen, crush the spleen with a 1 ml syringe pusher until no tissue fragments are visible and the fluid becomes turbid. Transfer the entire fluid to a 50 ml centrifuge tube and centrifuge at 600 × g for 3 minutes at room temperature.

Resuspend the cells in 400 ml of labeling buffer per spleen (approximately 1 × ¹⁰ cells per spleen). Add 100 μl of a biotinylated antibody cocktail (purchased from Miltenyi Biotec), mix gently, and incubate at 4°C in the dark for 5 minutes. Add 300 μl of labeling buffer and 200 μl of anti-biotin microbeads, and incubate at 4°C in the dark for 10 minutes. During this time, the magnet and magnet stand can be disinfected in a biosafety cabinet. After disinfection for 8 minutes, place the LS column in the center of the magnet and position it on the magnet stand, ensuring that a 15 ml centrifuge tube can fit underneath.

Add 1 ml of labeling buffer to the LS column to rinse, allowing the liquid to flow into the 15 ml centrifuge tube below. Once all the liquid has flowed out, transfer the T cell mixture to the LS column. Once all the liquid has flowed out, add 500 ml of labeling buffer to rinse the centrifuge tube and transfer it to the LS column. Then, add 2 ml of labeling buffer to rinse the LS column, allowing the collected liquid to flow into the centrifuge tube. Fill the centrifuge tube with PBS containing 2% FBS and centrifuge at 600 × g for 5 minutes at room temperature. Wash once with PBS containing 2% FBS.

First, pipette mouse T-activator CD3/CD28 activating magnetic beads (purchased from Thermo Fisher) at a concentration of 25 μl/1× ^10⁶ cells and rinse once with culture medium. T cells were then mixed with 1640 complete medium supplemented with 20 ng/ml IL-2 and 55 μM 2-mercaptoethanol to a concentration of (0.3-1) × ^10⁶ cells/ml. The T cell mixture and magnetic beads were mixed and plated at 1 ml per well in a 24-well plate for culture. After 48 hours of activation, OT-1 mouse ^CD8⁺ T cells were obtained.

Preparation Example 3. Preparation of CAR-T cells

CAR-T cells are obtained from healthy human whole blood CD8 ⁺ T cells and infected with human epidermal growth factor receptor-2 (Her2) lentivirus. The specific steps are as follows:

Use anticoagulant tubes to collect blood (either sodium heparin or EDTA), separate it immediately or store the healthy human peripheral blood at 4°C for transportation.

Add RosetteSep ^™ human CD8 ⁺ T cell enrichment mixture (purchased from Stem Cell) and antibodies directly to the blood collection tube at a rate of 50 μl/ml blood. After capping, gently invert upside down 3-5 times to mix thoroughly. Let it stand at room temperature for 20 minutes. During this time, you can prepare PBS containing 2% FBS. Then, according to the number of blood collection tubes and blood volume, use the corresponding separation tube (when the blood volume is less than 6 ml, use a 15 ml separation tube). Add ^{an appropriate amount of density gradient solution Ficoll to a 15ml centrifuge tube or SepMate™ tube} .

Gently mix the pre-prepared PBS containing 2% FBS with the blood in a 1:1 ratio (a 2:1-3:1 ratio is acceptable). Using a pipette, gently pipette the mixture 2-3 times to slowly layer it on top of the Ficoll solution, making sure not to disperse the Ficoll solution with the mixture. Secure the lid, carefully transfer the tube to a centrifuge, and centrifuge at 1200g for 20 minutes at room temperature.

After centrifugation, carefully transfer the tube to a biosafety cabinet. Use a pipette or Pasteur pipette to remove the middle buffy coat layer and transfer it to a new centrifuge tube. Fill the centrifuge tube with 1% FBS-containing PBS and wash once. Centrifuge at 600g for 10 minutes at room temperature. If red blood cells are present, lyse them and wash again. If not, wash again and centrifuge at 500g for 5 minutes at room temperature. Add X-VIVO15 complete medium containing 20ng/ml (100U/ml) interleukin-2 (IL-2) and count the cells. Then add 20ng/ml interleukin-7 (IL-7) and freeze at 1× ¹⁰⁶ cells/vial.

When needed immediately, first take activation magnetic beads (Dynabeads human T activator CD3/CD28 beads) at 25μl/1× ¹⁰⁶ cells and rinse once with culture medium. Then, T cells are prepared to a concentration of (0.3-1)× ¹⁰⁶ cells/ml with X-VIVO15 complete medium containing 20ng/ml IL-2. After mixing the T cell mixture and magnetic beads, add 1ml per well to a 24-well plate for culture.

Prepare a coating solution by adding 10 μl of 1 μg/ml Retronectin to 250 μl of PBS. Add the solution to a 24-well plate and incubate at room temperature for at least 30 minutes. Discard the coating solution and rinse once with PBS before use. After 24 hours of activation, transfer the T cells to a Retronectin-coated 24-well plate and continue incubation for another 24 hours.

Slowly add the corresponding CAR lentivirus directly to the T cell culture medium. After 6-8 hours of infection, gently change the medium without blowing away the attached T cells. Continue culturing for 24 hours before transferring to the culture flask.

The production process of CAR lentivirus is as follows:

PsPAX2 (Plasmid 12260) and pMD2.G (Plasmid 12259) plasmids were purchased from Addgene (MA, USA), and the Her2-specific CART plasmid (a gift from Professor Zhang Yi of the First Affiliated Hospital of Zhengzhou University) was owned by the laboratory.

Prepare the coating solution under sterile conditions according to the ratio of poly-lysine:gelatin = 1:4, add 5 ml of coating solution to each bottle and incubate overnight. Rinse once with PBS before use (or incubate at room temperature for 1 hour, then recover the coating solution and replace it with PBS for later use, and discard the PBS before use).

Thaw frozen 293T cells rapidly in a 37°C water bath, shaking repeatedly to accelerate thawing. Once all ice has melted, aseptically transfer the cells to a 15ml centrifuge tube pre-filled with 5ml of DMEM complete medium. Centrifuge at 1500 rpm for 5 minutes at room temperature. Discard the supernatant, resuspend the cells, and transfer them to a pre-coated T25 flask. Fill the flask with DMEM complete medium to a total of 10ml. Once the cells reach 90%-95% confluency, perform three passages.

Calculate the amount of plasmids and prepare a mixed solution. Lentivirus (LV) should be prepared in a ratio of 10:7:3: target plasmid, Her2-specific CART plasmid (50%), envelope plasmid, pSpAX2 (35%), and packaging plasmid, pMD2.G (15%). Add 10 μg of target plasmid, 7 μg of pSpAX2 plasmid, and 3 μg of pMD2.G plasmid to a T25 culture flask. Add the appropriate amount of OptiMEM medium to each T25 flask, calculated based on the amount of 250 μl of OptiMEM medium added to each T25 flask. Add the desired amount of plasmid to each 15 ml centrifuge tube and let stand for 5 minutes.

Prepare the transfection reagent mixture: add Opti MEM at 250 μl/bottle, then add the transfection reagent Viafect (total amount of plasmid ng: transfection reagent μl = 1:3) into a 15 ml centrifuge tube, blow evenly, and let it stand for 5 minutes.

After allowing to stand, transfer the transfection reagent mixture to the plasmid mixture at a volume of 250 μl/bottle, mix thoroughly, and let stand for 15 minutes. Gently aspirate the mixture and add 500 μl/bottle dropwise to the cells, gently shaking to mix. Label the mixture with the date and the name of the target plasmid.

6-8 hours after transfection, gently replace the medium with DMEM and add sodium butyrate (final concentration 1 mM), preparing it fresh for use. Harvest the virus 48 and 72 hours after the medium change. Concentrate the virus in a 100k concentrator tube, label it, and store it at -80°C.

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

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1×10 ⁶ LLC-OVA mouse lung cancer cells on the ventral side. Eight days after tumor inoculation, the mice were randomly divided into 4 groups (n=6 per group) based on tumor size and body weight. One group was intraperitoneally injected with blank nanoparticles (blank); one group was intraperitoneally injected with 50 μg/kg CD8-targeted G1P nanoparticles (G1P); one group was adoptively injected with 2×10 ⁶ OT-1 T cells via the tail vein and intraperitoneally injected with blank nanoparticles (OT1+blank); one group was adoptively injected with 2×10 ⁶ OT-1 T cells via the tail vein and intraperitoneally injected with 50 μg/kg CD8-targeted G1P nanoparticles (OT1+G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected every two days. After tumor inoculation Starting from the eighth day, the tumor size was measured every two days, and the tumor growth curve was recorded.

2. Experimental Results

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

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

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1× ¹⁰⁶ LLC-OVA mouse lung cancer cells in the ventral flank. 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 received intraperitoneal injection of blank nanoparticles (Blank); one group received intraperitoneal injection of 50 μg/kg CD8-targeted G1P nanoparticles (G1P); one group received tail vein transfer of 2× ¹⁰⁶ OT-1 T cells and intraperitoneal injection of blank nanoparticles (OT1+Blank); and one group received tail vein transfer of 2× ¹⁰⁶ OT-1 T cells and intraperitoneal injection of 50 μg/kg CD8-targeted G1P nanoparticles (OT1+G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected every two days. Long-term survival curves of the mice were recorded.

2. Experimental Results

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

Example 3. G1P promotes the formation of memory T cells.

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1× ¹⁰⁶ LLC-OVA mouse lung cancer cells in the ventral flank. Eight days after tumor inoculation, the 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. Mice were sacrificed 30 days after tumor inoculation, and lymph nodes were harvested, ground, filtered, and washed twice with PBS.

CD44 plays an important role in the formation and maintenance of long-term immune memory, helping T cells Central memory T cells rapidly initiate a response upon re-encountering the same antigen. CD62L is considered a marker for central memory T cells. These cells, with high CD62L expression, are able to rapidly localize to locations such as lymph nodes, enabling a rapid second response. Flow cytometry staining was used to measure the proportion of CD44 ⁺ CD62L ⁺ T cells in lymph nodes.

2. Experimental Results

Thirty days after tumor inoculation, that is, 22 days after the start of injection of blank nanoparticles or CD8-targeted G1P nanoparticles, the proportion of CD44 ⁺ CD62L ⁺ T cells in the lymph nodes of mice in the G1P treatment group was significantly increased compared with the blank group ( Figure 4 ).

Example 4. G1P promotes T cell infiltration.

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1× ^10⁶ LLC-OVA mouse lung cancer cells on the ventral flank. Eight days after tumor inoculation, the 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 CD8-targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected every two days. Thirty days after tumor inoculation, the mice were sacrificed, subcutaneous tumor tissue was excised and minced, and transferred to a 50ml centrifuge tube. 20ml of culture medium and 2mg/ml type IV collagenase were added and digested at 37°C, 120 rpm for 2-4 hours. After digestion, the tumor cell suspension was filtered. Immune-infiltrating lymphocytes were separated using Percoll, and the proportion of intratumoral CD8 ⁺ T cells was determined by flow cytometry.

2. Experimental Results

At 30 days after tumor inoculation, that is, 22 days after the injection of blank nanoparticles or CD8-targeted G1P nanoparticles, the proportion of CD8 ⁺ T cells in the tumor of mice in the G1P treatment group was significantly increased compared with that in the blank group ( Figure 5 ), indicating that G1P treatment promoted the tumor infiltration of T cells.

Example 5. G1P inhibits T cell exhaustion.

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1×10 ⁶ LLC-OVA mouse lung cancer cells on the ventral side. Eight days after tumor inoculation, the mice were randomly divided into two groups (n=6 per group) based on tumor size and body weight. One group received intraperitoneal injection of blank nanoparticles (blank); the other group received intraperitoneal injection of 50 μg/kg CD8-targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8-targeted G1P Nanoparticles were injected every two days. Mice were sacrificed 30 days after tumor inoculation, and subcutaneous tumor tissue was excised and minced. The tissue was transferred to a 50ml centrifuge tube and digested with 20ml of culture medium and 2mg/ml type IV collagenase at 37°C and 120rpm for 2-4 hours. After digestion, the tumor cell suspension was filtered to obtain the tumor cell suspension. Immune-infiltrating lymphocytes were separated using Percoll.

PD1, TIM3, and LAG3 are markers of T cell exhaustion and are highly expressed in exhausted T cells. Flow cytometry was used to detect the expression of PD1, TIM3, and LAG3, immune checkpoints on CD8 ⁺ T cells within the tumor.

2. Experimental Results

At 30 days after tumor inoculation, that is, 22 days after the injection of blank nanoparticles or CD8-targeted G1P nanoparticles, the expression of CD8 ⁺ T cell immune checkpoints PD1, TIM3, and LAG3 in the tumors of mice in the G1P treatment group was significantly decreased compared with the blank group ( Figure 6 ).

Example 6. G1P promotes cytokine production.

1. Experimental Procedure

C57BL/6J mice were subcutaneously inoculated with 1× ^10⁶ LLC-OVA mouse lung cancer cells on the ventral flank. Eight days after tumor inoculation, the 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 CD8-targeted G1P nanoparticles (G1P). Blank nanoparticles or CD8-targeted G1P nanoparticles were injected every two days. Thirty days after tumor inoculation, the mice were sacrificed, subcutaneous tumor tissue was excised and minced, and transferred to a 50ml centrifuge tube. 20ml of culture medium and 2mg/ml type IV collagenase were added and digested at 37°C, 120 rpm, for 2-4 hours. After digestion, the tumor cell suspension was filtered. Immune-infiltrating lymphocytes were separated using Percoll, and intratumoral CD8 ⁺ T cell expression of the cytokines TNF-α and IFN-γ was analyzed by flow cytometry.

2. Experimental Results

Thirty days after tumor inoculation, that is, 22 days after the start of injection of blank nanoparticles or CD8-targeted G1P nanoparticles, the expression of cytokines TNF-α and IFN-γ by CD8 ⁺ T cells in the tumors of mice in the G1P-treated group was significantly increased compared with the blank group ( Figure 7 ).

Example 7. G1P promotes CAR-T cell killing of tumors and inhibits tumor growth.

1. Experimental Procedure

Human melanoma tissue (F0 generation) was washed three times in PBS containing penicillin/streptomycin. The entire specimen was cut into tissues of approximately 5×5 mm in size. A single tumor tissue was implanted into the right flank of anesthetized NSG mice to produce the F1 generation. When the F1 generation tumor grew to 1 ^cm3 after implantation, it was excised and cut into multiple tissues (3×3 mm) and transplanted into new NSG mice to produce the next generation. After 3 generations, the mice were randomly divided into different groups, and after the F4 generation tumor grew to 5×5 mm, CAR-T cells (1× ¹⁰⁶ cells/mouse) were injected into the tail vein.

Mice were randomly divided into two groups (n=6 per group). One group received intraperitoneal injections of blank nanoparticles (Blank), while 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, and tumor growth curves were recorded.

2. Experimental Results

The tumor size in the G1P-treated group was significantly smaller than that in the blank group ( Figure 8 ), indicating that G1P treatment promoted CAR-T cell killing of tumors and inhibited tumor growth.

The results of the above examples show that:

CD8-targeted G1P nanoparticles were prepared and their targeting to CD8 ⁺ T cells was confirmed.

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

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 to kill tumors and inhibits tumor growth.

Compared to other drugs in clinical trials, G1P has a significant advantage in that it is an intermediate product of glycogen metabolism and is well tolerated by patients. G1P may promote glycogen metabolism, which in turn scavenges reactive oxygen species (ROS) by regulating the pentose phosphate pathway.

Claims (10)

Use of glucose-1-phosphate or a pharmaceutically acceptable salt thereof in preparing a medicament for treating tumors. Use of glucose-1-phosphate 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. 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, wherein the nanocarrier comprises poly(lactic-co-glycolic acid) (PLGA) and maleimide-poly(ethylene glycol) poly(lactic-co-glycolic acid) (PLGA-PEG-MAL); Preferably, the agent specifically targeting CD8 ⁺ T cells is an anti-CD8 antibody; Preferably, the surface of the nanocarrier is modified with streptavidin, and the reagent specifically targeting CD8 ⁺ T cells is a biotinylated anti-CD8 antibody; Preferably, the particle size of the nanoscale therapeutic agent is 50-250 nanometers, more preferably 100-200 nanometers. Use of the nanoscale therapeutic agent according to claim 3 in the preparation of a medicament for treating tumors. The use of the nanoscale therapeutic agent according to claim 3 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. The method 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, bile duct cancer, choriocarcinoma, pancreatic cancer, pediatric cancer, cervical cancer, ovarian cancer, breast cancer, bladder cancer, urothelial cancer, ureteral cancer, prostate cancer, seminoma, testicular tumor, head and neck tumor, head and neck squamous cell carcinoma, uterine cancer, endometrial cancer, thyroid cancer, lymphoma, myeloma, uterine carcinoma, endometrial cancer, thyroid cancer, lymphoma, myeloma, uterine carcinoma, uterine carcinoma, uterine duct cancer ... Tumor, osteoma, osteosarcoma, neuroblastoma, neuroblastoma, brain tumor, myeloma, astrocytoma, glioblastoma and glioma, preferably melanoma, lung cancer. The use according to any one of claims 1 to 2 and 4 to 5, wherein the drug is used in combination with another one or more treatment methods or therapeutic agents, the treatment methods are preferably radiotherapy, chemotherapy, immunotherapy, targeted therapy, and the therapeutic agent is preferably another agent for treating tumors; preferably, the treatment methods or therapeutic agents are administered simultaneously, sequentially or separately with the glucose-1-phosphate or a pharmaceutical composition containing the same. The use according to any one of claims 1 to 2 and 4 to 5, wherein the drug treats the tumor by: alleviating pathological symptoms and signs, preferably slowing 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; or enhancing the inhibitory effect of T cells on tumor growth. A method for preparing the nanoscale therapeutic agent according to claim 3, the method comprising: 1) Dissolve poly(lactic acid-co-glycolic acid) (PLGA) and poly(maleimide-polyethylene glycol) poly(lactic acid-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) ultrasonic emulsification and removal of dichloromethane and large particles; 4) Optionally, add streptavidin; 5) Optionally, a reagent that specifically targets CD8 ⁺ T cells is added. The reagent that specifically targets CD8 ⁺ T cells is preferably an anti-CD8 antibody, more preferably a biotinylated anti-CD8 antibody. A method for enhancing the anti-tumor activity of T cells in vitro, comprising the following steps: 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 according to claim 3, The T cells are preferably CD8 ⁺ T cells.