A method for the preparation of F-FDG labeled CART cells 18 A method for the preparation of F-FDG labeled CART cells

Abstract

This invention discloses a 18 This paper describes a method for preparing and applying 18F-FDG-labeled CAR-T cells, belonging to the field of cell labeling and molecular imaging technology. The preparation method includes five steps: cell preparation, glucose-free culture, radioactive labeling, washing and purification, and quality control. Optimization yielded a result requiring 1 hour of glucose-free culture and 960 μCi / 10⁻¹⁸ 18F-FDG. 6 The optimal labeling parameters for cells co-incubated for 1 hour were determined, and quality control standards were also defined. This invention solves the problems of low efficiency, affected activity, and poor stability in small-scale CAR-T cell labeling in existing technologies. After labeling, every 10... 6 The uptake value of each CAR-T cell reached 48.81 μCi, with a delabeling rate of only 18.01% after 1 hour. Cell viability was not significantly different from the control group, and the radiochemical purity reached 97.22%. The labeled cells can be used for PET imaging to clarify the in vivo distribution patterns of cells. The method is simple, low-cost, and has great potential for clinical translation. This invention also discloses the application of these labeled cells in PET imaging, providing an effective means for in vivo monitoring of low-dose CAR-T cell therapy.

Description

Technical Field

[0001] This invention relates to the field of cell labeling and molecular imaging technology, specifically to a... 18 Preparation method of F-FDG labeled CART cells. Background Technology

[0002] Cancer immunotherapy combats cancer by stimulating and enhancing the immune system. Chimeric Antigen Receptor T cell (CART) therapy is an emerging cancer immunotherapy. CART therapy involves modifying the patient's own T lymphocytes, loading them with receptors and co-stimulatory molecules that recognize tumor antigens, expanding them in vitro, and then reinfusing them into the patient to recognize and kill tumor cells. CART therapy has shown excellent efficacy in treating acute B-lymphoblastic leukemia (B-ALL) and large B-cell lymphoma and has been approved for marketing by the US FDA. In recent years, tumor immunotherapy has made remarkable progress, and its development prospects have attracted much attention, becoming one of the most watched events in the biomedical field in recent years.

[0003] Currently, studying the biological behavior of CAR-T cells in the "black box" after entering the body has become a popular research direction in molecular imaging. Cell labeling methods are broadly divided into indirect and direct cell labeling. Indirect cell labeling involves genetically manipulating cells through stable transfection of reporter genes, which induce protein expression. Imaging is then performed using imaging agents that specifically interact with these proteins. A key advantage of indirect cell labeling is that, ideally, the reporter gene protein is present throughout the cell's lifespan and is delivered during cell division. This allows for long-term in vivo imaging, providing information about cell proliferation, location, and viability throughout a subject's lifetime if properly calibrated. Despite these advantages, genetically manipulating cells for imaging is complex and technically challenging. For long-term imaging, repeated administration of tracers is required, posing significant difficulties for clinical translation and widespread adoption. In contrast, direct cell labeling is a simpler cell tracking method; any chemical reagent capable of penetrating cells or binding to the cell membrane can be used for radioactive cell labeling. Cells are typically labeled in vitro / outside of the body by incubating with a direct labeling agent and then injecting it into the subject. In vivo imaging can then be performed over time to assess biological behaviors such as cell distribution.

[0004] at present, 18 F-FDG has been applied to labeling studies of various cells, including leukocytes, granulocytes, and mesenchymal stem cells, but there are still many problems to be solved in CAR-T cell labeling:

[0005] 1. Low labeling efficiency: Existing studies mostly target 80-200×10⁻⁶ cells / year. 6 Large-scale, order-of-magnitude doses of CAR-T cells were used for labeling when applied to 10 6 When using small doses of CART cells, conventional labeling methods cannot achieve sufficient radioactivity to meet the requirements for clear PET imaging.

[0006] 2. Cell viability is affected: Centrifugation, washing and other operations during the labeling process, as well as the radiation effect of the radionuclide itself, may inhibit CART cell viability and affect its normal anti-tumor function;

[0007] 3. Poor labeling stability: 18 F-FDG-labeled cells have a high delabeling rate, and their short half-life (109.8 min) leads to a rapid decay of radioactivity during imaging, affecting imaging accuracy. Summary of the Invention

[0008] The present invention aims to solve the above-mentioned technical problems by providing a solution. 18 Preparation method of F-FDG labeled CART cells.

[0009] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0010] A sort of 18 The method for preparing F-FDG-labeled CART cells includes the following steps:

[0011] (1) Cell preparation: Human CD19-targeted CAR-T cells were obtained and cultured in RPMI-1640 medium containing 10% FBS. Before use, ensure that the cell viability is ≥85%, CD3+ cells are ≥80%, endotoxin is ≤1 EU / mL, mycoplasma test is negative, sterility test is negative, and CAR(+) cell transduction efficiency is ≥35%; adjust the CAR-T cells to (1±0.2)×10 6 Cell density per mL;

[0012] (2) Sugar-free culture: The CART cells prepared in step (1) were resuspended in sugar-free DMEM medium and cultured in a 37°C, 5% CO2 incubator for 1 h to deplete the endogenous glucose reserves of the CART cells.

[0013] (3) Radiolabeling: After sugar-free culture in step (2), CART cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was completely discarded, and the cells were gently resuspended in PBS containing calcium and magnesium ions at pH 7.4. The cell density was adjusted to (1 ± 0.2) × 10⁻⁶ cells / year. 6 / mL; per (1±0.2)×10 6Add 960±20 μCi / 100 μL of CART cells per mL. 18 F-FDG solution was placed in a 37℃, 5% CO2 incubator and incubated for 1 hour in a culture dish with a breathable lid. During incubation, the dish was gently shaken every 15 minutes to prevent cell sedimentation.

[0014] (4) Washing and purification: After incubation, immediately add 2 mL of ice-cold PBS pre-cooled to 4°C to terminate the process. 18 F-FDG uptake was performed, and the cells were quickly transferred to ice for handling. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was carefully discarded. The cells were washed three times with ice-cold PBS, replacing the centrifuge tube after each centrifugation to reduce residual radioactivity. Finally, the cell pellet was resuspended in PBS to obtain... 18 F-FDG labeled CART cells; labeled cells must be used for subsequent experiments within 2 hours.

[0015] (5) Quality control: Perform quality testing on the labeled CART cells obtained in step (4) to ensure that the cell viability is ≥90% and every 10 6 CART cell labeling rate ≥30 μCi, elution rate ≤20% after 1 hour, and radiochemical purity ≥97% immediately upon completion of labeling.

[0016] Preferably, the CART cells described in step (1) are cultured in a 37°C, 5% CO2 cell culture chamber. Cell morphology is observed daily under a microscope to ensure that the cells are round and permeable. The culture medium is gently pipetted until no cell clumps are present in a UV-sterilized laminar flow hood daily. Cell density and viability are monitored using an automated cell counter. Complete T-cell culture medium is replenished to maintain a cell density of (1±0.2)×10⁻⁶ cells / year. 6 / mL.

[0017] Preferably, the sugar-free DMEM medium is free of glucose, contains L-glutamine and sodium pyruvate, and is preheated to 37°C and pH adjusted to 7.2-7.4.

[0018] Preferably, the centrifugation operation in steps (3) and (4) is 1500 rpm for 5 minutes, and the centrifugation force is controlled at 290-310g. After centrifugation, the supernatant is gently aspirated to avoid disturbing the cell precipitation.

[0019] Preferably, the cell viability in step (5) is calculated as follows: cell viability = (experimental wells - blank wells) / (control wells - blank wells) × 100%; the elution rate is calculated as follows: elution rate = radioactivity of supernatant / (radioactivity of cell suspension + radioactivity of supernatant) × 100%; radiochemical purity is measured using a radioactive thin-layer scanner, and the labeling rate is detected using a gamma counter.

[0020] The present invention also provides 18The application of F-FDG-labeled CART cells in PET imaging: The preparation method yields labeled CART cells, which are then injected into the subject. PET dynamic scans are performed at 20 min, 40 min, 60 min, 120 min, 180 min, 240 min, and 300 min after injection to observe the distribution of CART cells in vivo.

[0021] When the subjects were mice, they were fasted and deprived of water for at least 4 hours, and anesthetized with 1.5-2.5% isoflurane. After anesthesia, they were fixed in a prone position and injected via the tail vein with 0.2 mL of a solution containing (1±0.2)×10⁻⁶ ppm. 6 / mL, with a radioactivity of 10-30 μCi 18 F-FDG labeled CART cells.

[0022] The PET dynamic scanning parameters are: slice thickness 2.0 mm, 5 min / bed, and whole-body images acquired in three-dimensional mode.

[0023] By employing the above method, the present invention has the following advantages:

[0024] 1. This invention uses 10 6 When using CART cells of the order of magnitude, the labeling method of this invention yields a cell uptake value of 48.81 μCi, compared to conventional methods (per 10 6 The cells were labeled with only 4-20 μCi, and the labeling activity was increased by 144%-1120%, which can meet the minimum activity requirements for clear PET imaging, and provides the possibility for in vivo monitoring of low-dose CAR-T cell therapy.

[0025] 2. The cell activity of the experimental group after labeling was not statistically different from that of the control group (p value < 0.05), which proves that 18F-FDG labeling does not significantly inhibit the activity of CART cells, ensuring that the labeled cells still have normal anti-tumor function and avoiding the impact of the labeling process on the treatment effect.

[0026] 3. The present invention achieves radiochemical purity of 97.22% immediately after labeling, with an elution rate of 18.01% within 1 hour and 22.01% within 2 hours, which is far lower than the labeling levels in similar studies (some studies achieved an elution rate of 70% within 3 hours). Furthermore, it clarifies the early distribution pattern of labeled cells in vivo (mainly in the lungs at 20 minutes, liver at 120 minutes, and spleen at 180 minutes), consistent with the in vivo distribution pattern of CAR-T cells over time, providing a quantitative basis for selecting the time window for clinical PET imaging.

[0027] 4. The 18F-FDG used in this invention is the most commonly used PET reagent in clinical practice and is readily available; the labeling parameters are standardized (1 hour of sugar-free culture, 1 hour of co-incubation, and 960 μCi / 10 of 18F-FDG).6 Cellular (Cellular) technology has a simple operation process, requires no special equipment, and significantly reduces the threshold and cost of clinical translation, making it easy to promote and apply.

[0028] 5. Compared with commonly used 89Zr and 68Ga labeling, 18F-FDG labeling has the advantages of simple operation, low cost of radionuclides, moderate half-life (109.8 min), and wide application, and has a broader application prospect in short-cycle CAR-T cell in vivo imaging research. Attached Figure Description

[0029] Figure 1 This is the invention 18 F-FDG activity versus cellular uptake diagram;

[0030] Figure 2 This is a diagram showing the changes in cell activity according to the present invention;

[0031] Figure 3 This is a graph showing the change in elution rate over time according to the present invention;

[0032] Figure 4 The present invention provides a radiochemically purified image obtained by R-TLC immediately after cell labeling is completed;

[0033] Figure 5 This is the invention 18 Imaging of F-FDG-labeled CART cells in mice.

[0034] Figure 6 yes 18 Flowchart of F-FDG-labeled CART cell manipulation and imaging. Detailed Implementation

[0035] The present invention will now be described in further detail with reference to the full text.

[0036] Combined with appendix Figures 1-5 ,A sort of 18 Methods for preparing F-FDG-labeled CART cells, and requirements for CART cells:

[0037] Human CD19-targeted CAR-T cells (cultured in RPMI-1640 medium containing 10% FBS); CAR-T cells were cultured in complete T-cell medium, ensuring cell viability ≥85% before use; CD3+ cells: ≥80%; endotoxin: ≤1 EU / mL; mycoplasma detection: negative; sterility test: negative; CAR-T (+) cells (transduction efficiency): ≥35%. CAR-T cells were cultured in a cell culture incubator (37℃, 5% CO2), and observed daily under a microscope to ensure round and permeable cell morphology. Daily, the culture medium was gently agitated with a pipette in a UV-sterilized laminar flow hood until no cell clumps remained. Cell density and viability were monitored using an automated cell counter. Complete T-cell medium was added as needed to maintain a cell culture density of (1±0.2)×10⁻⁶ cells / mL. 6 / mL.

[0038] Cell preparation and culture medium replacement steps:

[0039] CAR-T cells cultured to the logarithmic growth phase were harvested and accurately counted using an automated cell counter. The cell density was adjusted to (1 ± 0.2) × 10⁻⁶ cells / year. 6 / mL. Based on the principle that glucose-free culture can reduce competition for endogenous glucose within cells, previous experiments compared high-glucose, complete, low-glucose, and glucose-free media, finding that glucose-free media had the highest labeling efficiency. This protocol uses glucose-free DMEM medium (containing L-glutamine but no sodium pyruvate) for cell replacement. This medium needs to be preheated to 37°C and pH 7.2-7.4. During replacement, the original medium is removed by centrifugation at 1500 rpm for 5 minutes (approximately 300g), and the cells are gently resuspended in preheated glucose-free medium. Previous studies on glucose-free culture at 0, 30 min, 1 h, 1.5 h, and 2 h showed that as the glucose-free time increased, ... 18 F-FDG uptake gradually increased, reaching a plateau after 1 hour of glucose-free culture, and then slightly increased over time. This study selected a 1-hour culture incubator to deplete the cells' endogenous glucose reserves, maintaining the incubator at 37°C and 5% CO2.

[0040] Radioactive labeling procedure:

[0041] After incubation, transfer cells to 15mL centrifuge tubes and centrifuge at 1500rpm for 5 minutes (approximately 300g), thoroughly discarding the supernatant. Gently resuspend the cell pellet in PBS (containing calcium and magnesium ions, pH 7.4) and adjust the cell density to (1±0.2)×10⁻⁶ cells / mL. 6 / mL. Previous experiments showed that different doses... 18 F-FDG dosage and 10 6Co-incubation was conducted, and the results showed that starting from a dose of 160 μCi, cellular uptake increased with increasing dose, reaching a maximum at 960 μCi. Subsequently, with further increases in dose, cellular uptake decreased (e.g., ...). Figure 1 ), indicating differences 18 F-FDG dosage and 10 6 The pattern of cell uptake changes during co-incubation of several CAR-T cells was clarified. 18 The optimal dosage of F-FDG is 960 μCi.

[0042] This scheme yields (1±0.2)×10 6 / mLCART cell addition 18 F-FDG solution (960±20 μCi / 100 μL) was used to ensure a uniform distribution of radioactivity. Previous studies... 18 F-FDG was co-incubated for 30 min, 1 h, and 1.5 h, and the cell radioactivity was measured. Intracellular radioactivity peaked at 1 h, and the uptake value decreased after extending to 1.5 h. Therefore, this experiment selected... 18 The F-FDG solution co-incubates cells for 1 hour. During co-incubation, a breathable-lid culture dish should be used, and the incubator should be maintained at 37°C and 5% CO2. Gently shake the incubator every 15 minutes during incubation to prevent cell sedimentation.

[0043] Termination of reaction and washing and purification:

[0044] Immediately after 1 hour of incubation, add 2 mL of ice-cold PBS (pre-cooled to 4°C) to terminate the uptake, and quickly transfer to ice. Centrifuge at 1500 rpm for 5 minutes (approximately 300g), and carefully discard the supernatant (radioactive waste should be collected in a dedicated container). Repeat the washing with ice-cold PBS 3 times, replacing the centrifuge tube after each centrifugation to reduce residual radioactivity. Resuspend the final cell pellet in an appropriate amount of PBS, and sample for gamma counting to determine the labeling efficiency. Labeled cells must be used for subsequent experiments within 2 hours.

[0045] 18 F-FDG-labeled CART cell quality control requirements:

[0046] Cell viability was measured using a disposable cell counter to ensure a viability of at least 90% for CAR-T cells. Cell viability = (Experimental wells - Blank wells) / (Control wells - Blank wells) × 100%. Cell viability gradually decreases over time and with repeated centrifugation and washing (e.g., ...). Figure 2 ), Figure 2 This study aims to illustrate the changing trends of CART cell activity during the labeling process (at different time points and after multiple centrifugation and washing), and to verify the effect of the labeling method of this invention on cell activity.

[0047] Calculation of CART cell labeling rate: Radioactivity in the supernatant and intracellular fluid is measured using a radioactivity meter, and the result is calculated. 18 The labeling rate of F-FDG-labeled CART cells is required to be per 10 6 The CART cell labeling rate is not less than 30 μCi.

[0048] To measure the elution rate, labeled CAR-T cells were resuspended in complete T-cell culture medium and cultured in an incubator (37 ℃, 5% CO2). The cells were centrifuged at 1500 r for 5 min and washed three times with PBS. The elution rate was calculated, requiring it to be no higher than 20% after 1 hour. Elution rate = (Supernatant radioactivity / Cell suspension radioactivity) + Supernatant radioactivity × 100%. With increasing time, some labeled radionuclides eluted from the cells, with the most significant elution occurring at 30 min, gradually reaching equilibrium (e.g., ...). Figure 3 ), Figure 3 for 18 The changes in the unlabeling rate of CART cells after F-FDG labeling at different time points showed that the unlabeling process was most obvious at 30 min, and then tended to reach equilibrium.

[0049] Immediately after marking is completed, radiochemical purity (e.g., 100% radiometric purity) is measured using a radiometric thin-layer scanner. Figure 4 ), Figure 4 The image shows the radiochemical purity obtained by R-TLC immediately after cell labeling, indicating that the radiochemical purity was 97.22% immediately after labeling, demonstrating the specificity and stability of the labeling method of this invention.

[0050] CART-labeled animal experiments and PET imaging:

[0051] ① Mouse preparation: Fast the mice for at least 4 hours. Maintain anesthesia with 1.5-2.5% isoflurane. When the mice are drowsy and do not move when touched, fix them in a prone position with their limbs extended and fixed to a cardboard box.

[0052] 0.2 mL of 18F-FDG-labeled CART cells were injected via the tail vein (1 ± 0.2) × 10-1 6 / mL (10-30 μCi), i.e., start dynamic scanning for 1h, followed by static scanning for 10min at 2, 3, 4 and 5h respectively.

[0053] ② Scanning parameters:

[0054] PET images were acquired at a slice thickness of 2.0 mm, for 5 minutes per bed, using 3D mode to acquire whole-body images of the mouse (e.g., ...). Figure 5The results of whole-body PET imaging of mice at different time points after cell injection labeling were shown, clarifying the distribution pattern of cells in vivo (mainly in the lungs at 20 min, liver at 120 min, and spleen at 180 min).

[0055] This invention utilizes sugar-free culture to reduce competition for endogenous glucose within cells and enhance... 18 F-FDG transporter (GLUT1) expression; 18 After F-FDG enters the cell, it is phosphorylated by hexokinase and remains intracellularly. The appropriate co-incubation time balances uptake and radionuclide decay. This is achieved through optimizing sugar-free culture time, co-incubation time, and other factors. 18 The ratio of F-FDG dosage to cell number addresses the problem of insufficient activity of CART cell markers in small-scale cells.

[0056] This invention utilizes the scientific principle of controlling the radionuclide dosage and incubation time during the labeling process to avoid excessive radiation damage to cells and maintain normal cellular metabolic function. By screening for optimal labeling parameters, unnecessary processing steps are reduced, and it has been verified that the cell viability after labeling is not statistically different from that of the control group.

[0057] This invention can remove unbound free radicals through repeated washing. 18 F-FDG reduces non-specific radioactive interference; controlling the labeling time can reduce the impact of radionuclide decay on activity. By optimizing the post-labeling processing flow, high-level radiochemical purity labeling is achieved, and the labeling delabeling pattern is clarified.

[0058] Working principle: 18 F-FDG is a glucose analogue that can enter cells via the glucose transporter GLUT1 on the cell membrane and is phosphorylated by hexokinase to form glucose. 18 F-FDG-6-P, because it cannot participate in subsequent glucose metabolism, remains inside the cell, thus making the cell radioactive and detectable by PET imaging. This protocol maximizes cellular response to glucose by regulating the cell's metabolic state (glucose-free culture reduces endogenous glucose competition) and optimizing incubation conditions (time, dosage). 18 This allows for the uptake and retention of F-FDG while reducing the impact of radionuclide decay and delabeling on the labeling effect.

[0059] Operating steps (such as) Figure 6 ):

[0060] Cell preparation: Take cultured CART cells and adjust the cell concentration to (1±0.2)×10⁻⁶. ^6 / ml.

[0061] Sugar-free culture: The cells were placed in a sugar-free culture medium and cultured at 37°C and 5% CO2 for 1 hour.

[0062] 18 F-FDG incubation: After centrifugation to remove the culture medium, CART cells were resuspended in PBS solution, every 10... ^6 Add (960±20) μCi per ml of cells 18 F-FDG was further incubated at 37°C and 5% CO2 for 1 hour.

[0063] Washing and purification: Immediately after incubation, terminate the process with 2 ml of ice-cold PBS. 18 F-FDG uptake, followed by removal of unbound free FDG via centrifugation and washing. 18 F-FDG was used to wash the cells three times to obtain labeled CART cells.

[0064] Quality control: The radioactivity, radiochemical purity, and cell viability of labeled cells are tested to ensure they meet the requirements for PET imaging.

[0065] In vivo imaging: Labeled CAR-T cells were injected into the body, and dynamic PET scans were performed at 20, 40, 60, 120, 180, 240, and 300 minutes after injection to observe the cell distribution in vivo: at 20 minutes, the cells were mainly found in both lungs; at 120 minutes, the liver showed enhancement; and at 180 minutes, the spleen showed enhancement (e.g., ...). Figure 5 ).

[0066] An efficient labeling method for small-scale CART cells: targeting 10 6 Order-of-magnitude CAR-T cells were optimized to achieve sugar-free culture for 1 hour. 18 The optimal labeling parameters of 960 μCi F-FDG and co-incubation for 1 h solve the problem of insufficient cell labeling activity at low doses.

[0067] A strategy that balances labeling efficiency and cell viability: by controlling 18 By adjusting the F-FDG dosage and incubation time, high labeling activity can be achieved while ensuring that CART cell activity is not significantly inhibited, thus achieving a win-win situation for both labeling effect and cell function.

[0068] Quantitative evaluation and control methods for label stability: clarified 18 The radiochemical purity and labeling pattern of F-FDG-labeled CART cells (labeling rate of 18.01% at 1 hour and 22.01% at 2 hours) provide a quantitative basis for the selection of time windows for clinical PET imaging.

[0069] A marker-driven optimization mechanism based on cellular metabolic regulation reveals how glucose-free culture enhances GLUT1 expression and reduces endogenous glucose competition. 18 The mechanism of F-FDG uptake provides new insights into the metabolic regulation of cell markers.

[0070] Compared to commonly used directly labeled nuclides ( 89 Zr and 68 Ga marker), 18 F-FDG labeling has many advantages, including simple operation, low cost of nuclides, moderate half-life, and wide application. It is used in short-cycle research. 18 F-FDG has broad application prospects for in vivo imaging of direct labeling and tracking of CART cells.

[0071] Example 1: Preparation of 18F-FDG labeled CART cells.

[0072] 1. Materials preparation: Human CD19-targeted CAR-T cells (cultured in RPMI-1640 medium containing 10% FBS), complete T cell culture medium, sugar-free DMEM medium (glucose-free, containing L-glutamine, sodium pyruvate-free), PBS buffer (containing calcium and magnesium ions, pH 7.4), 18F-FDG solution (960 μCi / 100 μL), disposable cell counter, gamma counter, radioactive thin-layer scanner, centrifuge, 37℃, 5% CO2 incubator.

[0073] 2. Cell Culture and Preparation: CAR-T cells were cultured in complete T-cell culture medium and placed in a 37°C, 5% CO2 incubator. Cell morphology was observed daily under a microscope to ensure the cells were round and permeable. The culture medium was thoroughly agitated daily in a clean bench, and cell density and viability were monitored using an automated cell counter. Culture medium was replenished to maintain a cell density of 1.1 × 10⁻⁶ cells / day. 6 / mL. Cell quality was tested before use: cell viability 88%, CD3+ cells 82%, endotoxin 0.8 EU / mL, mycoplasma test negative, sterility test negative, CART(+) cell transduction efficiency 38%, which meets the requirements.

[0074] 3. Sugar-free culture: Take the above-mentioned CART cells, centrifuge at 1500 rpm for 5 minutes, remove the original culture medium, resuspend the cells in sugar-free DMEM medium preheated to 37℃ and pH 7.3, and adjust the cell density to 1.0 × 10⁶ cells / cm². 6 / mL, and incubated in a 37℃, 5% CO2 incubator for 1h.

[0075] 4. Radioactive labeling: After sugar-free culture, cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS to a concentration of 1.0 × 10⁻⁶. 6 / mL, add 960μCi of 18F-FDG solution to each 1mL of cell suspension, place in a breathable culture dish, and incubate at 37℃ and 5% CO2 for 1h, gently shaking once every 15 minutes.

[0076] 5. Washing and purification: After incubation, immediately add 2 mL of pre-chilled ice-cold PBS at 4°C to stop uptake, transfer to ice, centrifuge at 1500 rpm for 5 minutes, and discard the supernatant; repeat the washing with ice-cold PBS 3 times, replacing the centrifuge tube after each centrifugation, and finally resuspend the cell pellet with 1 mL of PBS.

[0077] 6. Quality Control: Cell viability was 92% as determined by a disposable cell counter; cell viability was measured every 10 cells using a gamma counter. 6 The labeling rate of CART cells was 48.81 μCi; the radiochemical purity was 97.22% as detected by a radiometric thin-layer scanner; after culturing the labeled cells for 1 hour, the unlabeling rate was 18.01%, all of which met the quality control requirements of this invention.

[0078] Example 2: 18 Application of F-FDG-labeled CART cells in PET imaging.

[0079] 1. Mouse preparation: Six-week-old Balb / c mice were selected. They were fasted and deprived of water for 4 hours before the experiment. They were anesthetized with 2.0% isoflurane. After the mice were put to sleep, they were fixed in a prone position on a cardboard box with their limbs extended and fixed.

[0080] 2. Cell injection: 0.2 mL of the cell culture prepared in Example 1 was injected via the tail vein. 18 F-FDG labeled CART cells, cell concentration 1.0 × 10⁻⁶ 6 / mL, radioactivity 20μCi.

[0081] 3. PET scan: Dynamic scanning was started immediately after injection for 1 hour, followed by static scanning for 10 minutes at 2 hours, 3 hours, 4 hours and 5 hours. The scanning parameters were: slice thickness 2.0 mm, 5 minutes / bed, and whole-body images of mice were acquired in three-dimensional mode.

[0082] 4. Results Analysis: The scanning results showed that CAR-T cells were mainly distributed in the lungs 20 minutes after injection, the liver began to show up after 120 minutes, and the spleen showed up after 180 minutes, which is consistent with the physiological distribution of CAR-T cells in the body after injection. The image clarity was good, and the dynamic distribution of cells in the body could be clearly tracked, proving that the labeled CAR-T cells prepared by this invention can meet the requirements of PET imaging.

[0083] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown throughout are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A kind 18 The method for preparing F-FDG-labeled CART cells is characterized by... Includes the following steps: (1) Cell preparation: Human CD19-targeted CAR-T cells were obtained and cultured in RPMI-1640 medium containing 10% FBS. Before use, ensure that the cell viability is ≥85%, CD3+ cells are ≥80%, endotoxin is ≤1 EU / mL, mycoplasma test is negative, sterility test is negative, and CAR(+) cell transduction efficiency is ≥35%; adjust the CAR-T cells to (1±0.2)×10 6 Cell density per mL; (2) Sugar-free culture: The CART cells prepared in step (1) were resuspended in sugar-free DMEM medium and cultured in a 37°C, 5% CO2 incubator for 1 h to deplete the endogenous glucose reserves of the CART cells. (3) Radiolabeling: After sugar-free culture in step (2), CART cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was completely discarded, and the cells were gently resuspended in PBS containing calcium and magnesium ions at pH 7.

4. The cell density was adjusted to (1 ± 0.2) × 10⁻⁶ cells / year. 6 / mL; per (1±0.2)×10 6 Add 960±20 μCi / 100 μL of CART cells per mL. 18 F-FDG solution was placed in a 37℃, 5% CO2 incubator and incubated for 1 hour in a culture dish with a breathable lid. During incubation, the dish was gently shaken every 15 minutes to prevent cell sedimentation. (4) Washing and purification: After incubation, immediately add 2 mL of ice-cold PBS pre-cooled to 4°C to terminate the process. 18 F-FDG uptake was performed, and the cells were quickly transferred to ice for handling. The cells were centrifuged at 1500 rpm for 5 minutes, and the supernatant was carefully discarded. The cells were washed three times with ice-cold PBS, replacing the centrifuge tube after each centrifugation to reduce residual radioactivity. Finally, the cell pellet was resuspended in PBS to obtain... 18 F-FDG labeled CART cells; labeled cells must be used for subsequent experiments within 2 hours. (5) Quality control: Perform quality testing on the labeled CART cells obtained in step (4) to ensure that the cell viability is ≥90% and every 10 6 CART cell labeling rate ≥30 μCi, elution rate ≤20% after 1 hour, and radiochemical purity ≥97% immediately upon completion of labeling.

2. The one according to claim 1 18 A method for preparing F-FDG-labeled CART cells, characterized in that: The CART cells described in step (1) were cultured in a 37°C, 5% CO2 cell culture chamber. Cell morphology was observed daily under a microscope to ensure that the cells were round and permeable. The culture medium was gently pipetted until no cell clumps were present in a UV-sterilized laminar flow hood. Cell density and viability were monitored using an automated cell counter. Complete T-cell culture medium was added to maintain a cell density of (1±0.2)×10⁻⁶ cells / year. 6 / mL.

3. The one according to claim 1 18 A method for preparing F-FDG-labeled CART cells, characterized in that: The sugar-free DMEM medium contains no glucose, L-glutamine, and no sodium pyruvate. It is preheated to 37°C and the pH is adjusted to 7.2-7.

4.

4. The one according to claim 1 18 A method for preparing F-FDG-labeled CART cells, characterized in that: The centrifugation operation in steps (3) and (4) is 1500 rpm for 5 minutes, and the centrifugation force is controlled at 290-310g. After centrifugation, the supernatant is gently aspirated to avoid disturbing the cell precipitation.

5. The one according to claim 1 18 A method for preparing F-FDG-labeled CART cells, characterized in that: The cell viability in step (5) is calculated as follows: cell viability = (experimental wells - blank wells) / (control wells - blank wells) × 100%; the elution rate is calculated as follows: elution rate = radioactivity of supernatant / (radioactivity of cell suspension + radioactivity of supernatant) × 100%; radiochemical purity is measured using a radioactive thin-layer scanner, and the labeling rate is detected using a gamma counter.

6. A kind 18 The application of F-FDG-labeled CART cells in PET imaging is characterized by... Labeled CART cells were obtained using any one of the preparation methods described in claims 1-5, and injected into the subject. PET dynamic scans were performed at 20 min, 40 min, 60 min, 120 min, 180 min, 240 min, and 300 min after injection to observe the distribution of CART cells in vivo.

7. The application according to claim 6, characterized in that: When the subjects were mice, they were fasted and deprived of water for at least 4 hours, and anesthetized with 1.5-2.5% isoflurane. After anesthesia, they were fixed in a prone position and injected via the tail vein with 0.2 mL of a solution containing (1±0.2)×10⁻⁶ ppm. 6 / mL, with a radioactivity of 10-30 μCi 18 F-FDG labeled CART cells.

8. The application according to claim 6, characterized in that: The PET dynamic scanning parameters are: slice thickness 2.0 mm, 5 min / bed, and whole-body images acquired in three-dimensional mode.

Images