Aptamer for specifically targeting CAR-T cells, enrichment method, application and product

Abstract

The invention belongs to the field of biological detection, and particularly relates to a nucleic acid aptamer for specifically targeting CAR-T cells, an enrichment method, application and a product. The nucleic acid aptamer has a sequence as shown in SEQ ID NO. 1. According to the invention, the CAR-T cell is used as a screening target, and the nucleic acid aptamer with specificity and affinity to the CAR-T cell is obtained. The aptamer can be used for construction of a CAR-T cell imaging probe, specific recognition and combination with CAR-T cells, and monitoring of the CAR-T cells at tumor tissues is achieved. The invention further designs a complementary sequence of the nucleic acid aptamer, and the CAR positive T cells are enriched in a traceless manner in an oligonucleotide complementary manner.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a nucleic acid aptamer specifically targeting CAR-T cells and an enrichment method, application and product. Background Art

[0002] Chimeric antigen receptor T cells (CAR-T) therapy uses genetic engineering to modify synthetic CARs on the surface of T cells to target tumors and eliminate cancer cells. CAR-T cells have achieved results in the treatment of a variety of hematological malignancies. However, the response of patients receiving CAR-T cell therapy is not consistent, and many cancer patients do not respond to CAR-T cell therapy or experience subsequent relapse. As a "living" drug, the problems of monitoring the in vivo distribution of CAR-T cells and evaluating their pharmacokinetic properties remain unsolved. Diagnostic technology that directly monitors the localization and proliferation of CAR-T cells at the tumor site will help optimize personalized tumor treatment plans.

[0003] Currently, there is no standardized method to monitor the distribution and targeting efficacy of injected CAR-T cells in vivo. Clinical researchers use various strategies to evaluate the activity of injected CAR-T cells, such as flow cytometry, immunohistochemistry, and qPCR of peripheral blood samples, or biopsies from active CAR-T cell sites, such as bone marrow, tumors, and lymph nodes. However, in clinical trials, repeated invasive biopsies are impractical and insufficient to evaluate the therapeutic response of multiple metastatic sites. Therefore, it is necessary to construct specific recognition probes for CAR-T cells and combine molecular imaging technology to monitor the biological distribution, survival and proliferation of CAR-T cells in tumor tissues, as well as whether there are off-target effects. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a nucleic acid aptamer specifically targeting CAR-T cells and an enrichment method, application and product.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] A nucleic acid aptamer specifically targeting CAR-T cells, wherein the nucleic acid aptamer has a sequence shown in SEQ ID NO.1.

[0007] SEQ ID NO.1:

[0008] 5'-TTCAGCACTCCACGCATAGCACGCTGCCGTGTGCTGCATGTGGTCGTGTGTCCGTGCCTATGCGTGCTACCGTGAA-3'.

[0009] The nucleic acid aptamer is modified with a functional group or molecule; preferably, the functional group or molecule is a fluorescent substance, a nanoluminescent material, biotin, a radionuclide, or an enzyme marker.

[0010] The present invention also includes a method for enriching the CAR-T cells, using a complementary sequence RA to release the CAR-T cells recognized and bound by the nucleic acid aptamer sequence; the RA has a sequence shown in SEQ ID NO.2;

[0011] SEQ ID NO. 2: 5'-TTCACGGTAGCACGCATAGGCAC-3'.

[0012] The present invention also includes an application of the nucleic acid aptamer specifically targeting CAR-T cells, which is used to detect or track CAR-T cells.

[0013] The present invention also includes a product for detecting or tracking CAR-T cells, including the nucleic acid aptamer specifically targeting CAR-T cells.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention uses CAR-T cells as screening targets and obtains nucleic acid aptamers with specificity and affinity for CAR-T cells. The aptamer can be used to construct a CAR-T cell imaging probe, specifically recognize and bind to CAR-T cells, and monitor CAR-T cells in tumor tissues. The present invention also designs a complementary sequence of the nucleic acid aptamer, and uses oligonucleotide complementation to enrich CAR-positive T cells without traces.

[0016] In summary, the present invention has obtained a new CAR-T cell aptamer, which provides an efficient, accessible and inexpensive alternative tool to enrich, monitor and reposition CAR-T cells for potential clinical-scale cell therapy applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A is the flow cytometry characterization of different rounds of aptamer enrichment libraries and CAR-T cells and Mock-T cells during the Cell-SELEX process;

[0018] Figure 1 B is the DNA primary sequence comparison analysis of the top 30 sequences with the highest number of repetitions in the sequencing results after high-throughput sequencing of the 11th round of evolutionary library using DNAMAN;

[0019] Figure 1C is the flow cytometry characterization of candidate nucleic acid aptamers, CAR-T cells, and Mock-T cells after sequencing;

[0020] Figure 1 D is the analysis of the apparent dissociation constant (Kd) of nucleic acid aptamer A3 binding to CAR-T cells;

[0021] Figure 1 E is the confocal fluorescence imaging characterization of nucleic acid aptamer A3, CAR-T cells, and Mock-T cells (blue and green signals represent DAPI and FAM-labeled aptamer A3, respectively. Scale bar: 20 μm);

[0022] Figure 1 F is the binding specificity analysis of nucleic acid aptamer A3 and different cells;

[0023] Figure 2 AB is the stability of aptamer A3 in human serum;

[0024] Figure 2 C is the in vivo fluorescence imaging of nucleic acid aptamer A3-Cy5 injected into Nalm6 tumor-bearing mice with or without CAR-T cells through tail vein.

[0025] Figure 2 D is the in vitro fluorescence imaging of nucleic acid aptamer A3-Cy5 injected into Nalm6 tumor-bearing mice with or without CAR-T cells through tail vein.

[0026] Figure 2 E is an immunofluorescence staining image of tumor tissue after tail vein injection of nucleic acid aptamer A3-Cy5 (blue: DAPI; red: aptamer A3-Cy5, green: anti-CD8-FITC. Scale bar: 100 μm);

[0027] Figure 3 A is a schematic diagram of traceless enrichment of CAR-T cells by nucleic acid aptamer A3;

[0028] Figure 3 B is the positive rate of CAR-T cells in the mixed cell population of CAR-T cells and Mock-T cells;

[0029] Figure 3 C is the minimum free energy (MFE) secondary structure of modified aptamer A3 predicted by NUPACK software. The orange line indicates a 23nt region where a complementary reversal agent (RA) is designed to anneal. The right figure shows the predicted MFE secondary structure after the displacement of aptamer A3 and RA chain;

[0030] Figure 3 D is the positive rate of CAR-T cells after aptamer A3 captured CAR-T cells;

[0031] Figure 3 E is the flow cytometry analysis of the fold change in the proportion of CAR-positive cells before and after CAR-T cell sorting using nucleic acid aptamer A3. DETAILED DESCRIPTION

[0032] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and the best embodiments.

[0033] The reagents configured in the present invention are:

[0034] Washing Buffer: Weigh 0.225 g glucose and 0.050825 g magnesium chloride hexahydrate and dissolve them in 50 mL DPBS. Store at 4°C for 1 month.

[0035] Binding Buffer: Weigh 0.05 g BSA, add 10 mL Washing Buffer, then add 100 μL tRNA (100 mg / mL), fully dissolve, and store at 4°C for 1 month.

[0036] Instruments used in the present invention:

[0037] Electronic balance (Zhuojin, Shanghai), centrifuge (Thermo, MicroCL 21R, USA), flow cytometer (GuavaeasyCyte, USA), laser confocal microscope (Leica Stellaris 5, Germany), fluorescence inverted microscope (Leica, DMI300B, Germany), gel imager (Bio-Rad USA), IVIS Lumina S5 small animal living imaging system (PerkinElmer Health Sciences, MA, USA).

[0038] Example 1: Screening of CAR-T cell nucleic acid aptamers using Cell-SELEX technology

[0039] (1) The design of random library and primers is as follows:

[0040] Random library: 5'-TTCAGCACTCCACGCATAGC-36N-CCTATGCGTGCTACCGTGAA-3', where 36 Ns represent 36 random nucleotides;

[0041] Forward primer: 5′-FAM-TTCAGCACTCCACGCATAGC-3′;

[0042] Reverse Primer:

[0043] 5'-AAAAAAAAAAAAAAAAAA-Spacer18-TTCACGGTAGCACGCATAGG-3'.

[0044] (2) Cell culture: CAR-T cells and Mock-T cells were cultured in LONZA X-VIVO 15 complete medium containing 10% fetal bovine serum (Hyclone, USA). 200 U / mL IL-2, 10 ng / mL IL-7, and 10 ng / mL IL-15 were added to the medium. The cells were cultured in a 5% CO 2 Culture in a humidified incubator.

[0045] (3) Cell-SELEX screening (rounds 1-3): 2 nmol of random library was dissolved in 200 μL of DPBS, mixed and denatured at 95°C for 10 min, cooled on ice for 5 min, and placed at 4°C for standby use. CAR-T cells were cultured in a T25 culture flask. After the cell density reached 90%, the cells were washed twice with Washing Buffer, and the suspended cells were centrifuged at 1100 rpm for 5 min. After counting the cells, they were resuspended in Binding Buffer, and the random library was added to the CAR-T cell suspension and incubated on a shaker at 4°C for 1 h. After the incubation, the cells were washed twice with Washing Buffer, collected and resuspended in Binding Buffer, heated at 95°C for 10 min, cooled on ice for 5 min, and centrifuged at 14000 rpm for 5 min at 4°C. The supernatant was recovered, a portion was used for PCR amplification, and the rest was stored at -20°C for standby use.

[0046] (4) PCR amplification: 500 μL system

[0047] Reaction components Volume (μL) Random Library 100 Q5 enzyme 3 Forward primer (100 μM) 2.5 Reverse primer (100 μM) 2.5 dNTPMIX (10 μM) 10 5×Q5buffer 100 dd water 282

[0048] PCR program settings: 10-25 cycles (95°C for 30 s, 60°C for 30 s, 72°C for 30 s), 72°C for 3 min.

[0049] (5) Preparation of ssDNA: Since the reverse primer used has an extended nucleic acid fragment, the short-chain and long-chain separation method is used to prepare single-stranded DNA.

[0050] First, prepare polyacrylamide denatured gel, take 9ml of denatured gel mixed liquid, add 64μL ammonium persulfate and mix well, then add 8μL TEMED and mix well, pour it into the assembled gel plate, insert a suitable electrophoresis comb to assemble the prepared gel plate, set the current to 300mA, and pre-electrophoresis for 30 minutes. During the pre-electrophoresis process, take the concentrated PCR product, add an equal volume of 2×TBE urea loading buffer in the kit, boil in water bath for 10min, centrifuge instantly, and load the sample for electrophoresis immediately. After the electrophoresis is completed, take out the denatured gel and cut the gel under 254nm ultraviolet light. Put the cut gel into a gel centrifuge tube, centrifuge at 12000rpm for 2min, add 1mL DPBS, boil in water bath for 10min, centrifuge at 12000, 2min, and aspirate the supernatant into a 15mL centrifuge tube. Add 5.5 times the volume of n-butanol to a 15 mL centrifuge tube, centrifuge at 9000 rpm for 5 min, and the solution will be separated into layers. Aspirate and discard the upper clear liquid, transfer the lower single-stranded library to a 2 mL centrifuge tube, and use a micro-nucleic acid dialysis membrane for desalting. After dialysis, use UV260 to calibrate the ssDNA content, and put the ssDNA into the next round of screening.

[0051] (6) Cell-SELEX screening (rounds 4-11): Denature the single-chain prepared in the previous round and resuspend it in the binding buffer. In order to further improve the affinity and specificity of the nucleic acid aptamer, the screening pressure was gradually increased during the screening process. The screening conditions were set as shown in Figure 1, including: reducing the number of CAR-T cells, increasing the number of Mock-T cells, increasing the number and time of washing, and increasing the BSA concentration of the Binding Buffer.

[0052] Table 1 shows the screening conditions of nucleic acid aptamers in different rounds during Cell-SELEX;

[0053]

[0054] (7) Flow cytometry analysis of nucleic acid library enrichment: CAR-T cells and Mock-T cells cultured in T25 cell culture flasks were taken; the initial library, the single-chain library obtained by the 5th, 7th, 9th, and 11th rounds of screening were taken, dissolved in 200 μL Binding Buffer, the final concentration was 250 nmol / L, heated at 95°C for 10 min, cooled on ice for 5 min, added to CAR-T cells and Mock-T cells respectively, placed on ice, protected from light, and incubated on a horizontal shaker for 40 min. After incubation, the cells were washed twice with Washing Buffer, and the cells were resuspended into a single cell suspension. The fluorescence value of each group of cells after binding to the library was detected by flow cytometry, and the initial library binding to the cells was used as a negative control.

[0055] Flow cytometry was used to detect the binding of the library to the cells during the screening process. Figure 1 As shown in A, as the library screening cycle progresses, compared with the control group, the fluorescence intensity of the evolved library binding to CAR-T cells detected by flow cytometry has a significant shift, while the fluorescence intensity of the evolved library binding to Mock-T cells has no shift.

[0056] (8) High-throughput sequencing: After selecting the nucleic acid library from the 11th round of screening for PCR amplification, the product was sent to Shanghai Biotechnology Co., Ltd. for high-throughput sequencing. Figure 1 As shown in B, based on the sequence repetitiveness in the sequencing results, DNAMAN was used to perform DNA primary sequence comparison analysis on the top 30 sequences with the highest number of repetitions in the sequencing results. Based on the sequence homology, we selected 8 candidate aptamers for binding studies with CAR-T cells and Mock-T cells. Figure 1 As shown in Figure C, compared with the control group, the fluorescence intensity of aptamer A3 binding to CAR-T cells detected by flow cytometry had the largest shift, while the fluorescence intensity of aptamer A3 binding to Mock-T cells had no obvious shift. Therefore, aptamer A3 (having the sequence shown in SEQ ID NO.1, 5'-TTCAGCACTCCACGCATAGCACGCTGCCGTGTGCTGCATGTGGTCGTGTGTCCGTGCCTATGCGTGCTACCGTGAA-3') was selected for subsequent studies.

[0057] (9) Determination of the apparent Kd value of aptamer A3 binding to CAR-T cells: Take a bottle of CAR-T cells cultured in a T25 bottle; select FAM-labeled nucleic acid aptamer A3 with a concentration gradient of 1nmol / L, 2nmol / L, 5nmol / L, 10nmol / L, 20nmol / L, 50nmol / L, 100nmol / L, and 200nmol / L. After adding the nucleic acid aptamer A3 of each concentration gradient to the CAR-T cells, place it on ice and incubate it on a horizontal shaker for 40 minutes away from light. After the incubation, wash the cells twice with Washing Buffer, resuspend the cells into a single cell suspension, and detect the binding fluorescence value of each group of nucleic acid aptamer A3 and CAR-T cells by flow cytometry. Finally, use SigmaPlot10.0 software for analysis, according to the fitting formula: Y=B max X / (Kd+X), and the fitting curve and Kd value of the aptamer are obtained. Figure 1 As shown in D, the equilibrium dissociation constant (Kd value) between aptamer A3 and CAR-T cells is 16.5±4nM, indicating that aptamer A3 has a strong affinity with CAR-T cells.

[0058] (10) Cell imaging of nucleic acid aptamer A3: Take the CAR-T cells and Mock-T cells cultured in a T25 culture flask, wash the cells twice with Washing Buffer, add FAM-labeled nucleic acid aptamer A3 at a final concentration of 250nmol / L, place on ice, protect from light, and incubate on a horizontal shaker for 40 minutes. After incubation, wash the cells twice with Washing Buffer, centrifuge at 1100rpm for 5 minutes, collect the cell samples in a 1.5mL centrifuge tube, add 400μL of 4% paraformaldehyde fixative, slowly resuspend the cells, and let stand for 10 minutes. Centrifuge to remove the fixative, wash the cells twice with Washing Buffer, then drop the cells onto a glass slide, drop DAPI stain on the cell suspension, dry it slightly, and cover it with a clean coverslip. Observe using a laser confocal scanning microscope. Figure 1 As shown in E, aptamer A3 aggregates around CAR-T cells and has affinity for them, but has no binding properties to Mock-T cells.

[0059] (11) Characterization of the specificity of nucleic acid aptamer A3: Nalm6 cells and U266 cells were cultured in RPMI 1640 medium containing 10% heat-inactivated fetal bovine serum. RAW264.7 cells and GL-261 cells were cultured in DMEM medium containing 10% fetal bovine serum. All cells were cultured in a humidified incubator containing 5% carbon dioxide. Figure 1 As shown in F, flow cytometry was used to detect the fluorescence intensity of aptamer A3 binding to the cells. Compared with the random library control group, there was no obvious fluorescence shift between aptamer A3 and the cells. Therefore, aptamer A3 has good specificity for CAR-T cells.

[0060] Example 2: Imaging of nucleic acid aptamer A3-Cy5 for tracking CAR-T cells at the tumor site

[0061] (1) Characterization of the stability of nucleic acid aptamers in human serum: Nucleic acid aptamer A3 was incubated at 37°C for 1 h in human serum at 0%, 20%, 50%, and 100% concentrations. Nucleic acid aptamer A3 was incubated in human serum at 20% concentration for 2 h, 4 h, 8 h, 12 h, and 24 h. After the incubation, the relative content of nucleic acid aptamers was detected by 3% agarose gel electrophoresis. After the electrophoresis, the image was displayed using a gel imager (Bio-Rad USA) and analyzed by Image Lab (Bio-Rad USA).

[0062] like Figure 2 As shown in AB, the aptamer A3 band gradually dimmed with the increase of serum concentration, but the A3 band could still be observed at 100% serum concentration and remained stable after incubation with 20% serum concentration for 12 h.

[0063] (2) Fluorescence imaging of CAR-T cells by nucleic acid aptamer A3-Cy5 in Nalm6 tumor-bearing mouse model: Severely immunodeficient female mice (NOG) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. 2x10 6 Nalm6 cells were implanted in the subcutaneous part of the right thigh of mice to establish a Nalm6 tumor-bearing mouse model. All animal experiments were conducted in accordance with the Principles of Laboratory Animal Care of the People's Republic of China and the Guide for the Care and Use of Laboratory Animals of Xuzhou Medical University, China (Permit No. 202406T003). Each tumor-bearing mouse in the experimental group was injected with 2×10 6 CAR-T cells were injected into the mice via the tail vein 30 min after the injection of CAR-T cells. Mice with random sequences labeled with Cy5 and without CAR-T cells were used as the control group. All mice were anesthetized with isoflurane, and the fluorescence signals of CAR-T cells in vivo were collected in the tumor area 30 min after the injection of aptamer A3-Cy5 using the IVIS Lumina S5 small animal imaging system. The mice were then euthanized, and ex vivo fluorescence imaging was performed after dissection of the heart, liver, spleen, lung, kidney, and tumor. Fluorescence images were analyzed using the Living Image software (Perkin Elmer, MA, USA). Tumors were sent to Seville Biotechnology Co., Ltd. for CD8 antibody staining, sections were made, and images were acquired by fluorescence microscopy.

[0064] like Figure 2 As shown in Figure C, when the nucleic acid aptamer A3-Cy5 was injected into mice containing CAR-T cells, obvious fluorescence signals were visible in the tumor area. In contrast, after the random sequence injection labeled with Cy5, no obvious fluorescence signals were observed in the tumor area. We introduced the Cy5-labeled aptamer A3 into a tumor mouse model lacking CAR-T cells. Initially, the fluorescence signal was evenly distributed, but 50 minutes after the injection of the aptamer A3-Cy5, no obvious fluorescence signal was detected in the tumor area. In addition, as Figure 2 As shown in D, the results of in vitro imaging of mice with resected organs were similar to those of in vivo imaging. After injection of aptamer A3-Cy5, the fluorescence signal of the dissected tumor was significantly higher than that of the control group. Figure 2 As shown in E, CD8+ T cells are the main cells mediating antigen-specific immune responses in CAR-T cell therapy. Staining of tumor tissue sections with CD8 antibodies further demonstrated that CAR-T cells can be tracked in vivo by aptamer A3-Cy5.

[0065] Example 3: Traceless enrichment of CAR-T cells based on nucleic acid aptamer A3:

[0066] (1) Modification and synthesis of nucleic acid aptamer A3-biotin and complementary sequence RA: As shown in Figure 2, the nucleic acid aptamer sequences were ordered from Shanghai Shenggong Biotechnology Co., Ltd.

[0067] Table 2 Aptamer A3 and complementary sequence RA

[0068]

[0069] (2) Coupling of nucleic acid aptamer A3-biotin and streptavidin magnetic beads: The biotin-labeled aptamer A3 was incubated with streptavidin magnetic beads at 4°C overnight.

[0070] (3) Preparation of mixed cell populations: Take the CAR-T cells and Mock-T cells cultured in T25 culture flasks, count them using a cell counting plate, and mix the CAR-T cells and Mock-T cells in a 1:1 ratio.

[0071] (4) Detection of CAR-T cell positivity in mixed cell population: The constructed mixed cell population was incubated with Protein-L antibody (containing biotin) at 4°C for 30 min. After centrifugation and washing twice with Washing Buffer, Anti-APC streptavidin was added and incubated in the dark for 30 min. After centrifugation and washing twice with Washing Buffer, flow cytometry was performed. The test results are as follows: Figure 3 As shown in B.

[0072] (5) Nucleic acid aptamer A3 seamlessly enriches CAR-T cells: Figure 3 As shown in A, it is a schematic diagram of the traceless enrichment of CAR-T cells using aptamer A3. The biotin-labeled aptamer A3 is pre-loaded on the streptavidin-labeled magnetic beads and incubated with different cell components. After the incubation, the CAR-T cells enriched by magnetic force are added with 10 times the complementary sequence RA of A3-biotin. Shake in a shaker at 37°C for 1 hour. The CAR-T cells are separated from A3-biotin by oligonucleotide complementation, and the magnetic beads are separated from the cells using a magnetic stand. Figure 3 C shows the minimum free energy (MFE) secondary structure of the modified aptamer A3 predicted by NUPACK software. The orange line indicates a 23nt region where a complementary reversal agent (RA) is designed to anneal. The right figure shows the predicted MFE secondary structure after chain displacement of aptamer A3 and RA;

[0073] (6) Detection of CAR-T cell positivity after traceless enrichment with aptamer A3: After magnetic separation of the mixed cell population, the supernatant containing CAR-T cells was collected and incubated with Protein-L antibody (containing biotin) at 4°C for 30 min. After centrifugation and washing twice with Washing Buffer, Anti-APC streptavidin was added and incubated in the dark for 30 min. After centrifugation and washing twice with Washing Buffer, flow cytometry was performed. The test results are as follows: Figure 3 As shown in D.

[0074] (7) Data analysis: Figure 3 As shown in E, flow cytometry analysis showed that the CAR-T cell positivity rate increased by 1.6 times after the mixed cell population was seamlessly enriched with nucleic acid aptamer A3.

[0075] The present invention uses CAR-T cells as screening targets and obtains nucleic acid aptamers with specificity and affinity for CAR-T cells. The aptamer can be used to construct a CAR-T cell imaging probe, specifically recognize and bind to CAR-T cells, and monitor CAR-T cells in tumor tissues. The present invention also designs a complementary sequence of the nucleic acid aptamer, and uses oligonucleotide complementation to enrich CAR-positive T cells without traces.

[0076] In summary, the present invention has obtained a new CAR-T cell aptamer, which provides an efficient, accessible and inexpensive alternative tool to enrich, monitor and reposition CAR-T cells for potential clinical-scale cell therapy applications.

[0077] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nucleic acid aptamer specifically targeting CAR-T cells, characterized in that: The nucleic acid aptamer has the sequence shown in SEQ ID NO.

1.

2. The nucleic acid aptamer specifically targeting CAR-T cells according to claim 1, characterized in that: SEQ ID NO.1: 5'-TTCAGCACTCCACGCATAGCACGCTGCCGTGTGCTGCATGTGGTCGTGTGTCCGTGCCTATGCGTGCTACCGTGAA-3'.

3. The nucleic acid aptamer specifically targeting CAR-T cells according to claim 1, characterized in that: The nucleic acid aptamer is modified with a functional group or molecule; preferably, the functional group or molecule is a fluorescent substance, a nanoluminescent material, biotin, a radionuclide, or an enzyme marker.

4. A method for enriching CAR-T cells according to any one of claims 1 to 3, characterized in that: The complementary sequence RA is used to release the CAR-T cells recognized and bound by the nucleic acid aptamer sequence; the RA has the sequence shown in SEQ ID NO.

2.

5. The method for enriching CAR-T cells according to claim 4, characterized in that: Complementary sequence SEQ ID NO. 2: 5'-TTCACGGTAGCACGCATAGGCAC-3'.

6. A use of a nucleic acid aptamer specifically targeting CAR-T cells according to any one of claims 1 to 3, characterized in that: Applicable to detecting or tracking CAR-T cells; preferably used for imaging tracking CAR-T cells at tumor sites.

7. A product for detecting or tracking CAR-T cells, characterized in that: A nucleic acid aptamer specifically targeting CAR-T cells comprising any one of claims 1 to 3.

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