Targeted uPAR TRuC-T cells expressing IL-7 and PF4 for enhanced anti-tumor activity and uses

Abstract

This invention relates to the field of biotechnology, specifically to a TruC-T cell that secretes and expresses IL-7 and PF4 to enhance anti-tumor activity and targets uPAR, and its application. Interleukin-7 (IL-7) can effectively promote the development, survival, and proliferation of T cells, while platelet factor 4 (PF4 / CXCL4) can regulate the immune response and chemotactically attract leukocytes to specific sites; the synergistic effect of the two can enhance the anti-tumor function and persistence of T cells. Meanwhile, urokinase-type plasminogen activator receptor (uPAR) is highly expressed in various solid tumors and hematological malignancies, making it an ideal target for tumor therapy; TruC-T cells targeting uPAR can precisely act on tumor tissue, and combined with the immune-enhancing effects of IL-7 and PF4, can significantly improve the efficacy of anti-tumor immune responses, prolong the duration of therapeutic effects, and reduce tumor recurrence.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a TRuC-T cell that secretes and expresses IL-7 and PF4 to enhance anti-tumor activity and targets uPAR, and its application. Background Technology

[0002] Adoptive cellular immunotherapy (ACT), particularly chimeric antigen receptor T-cell (CAR-T) therapy, has achieved revolutionary success in the treatment of hematologic malignancies. However, CAR-T therapy faces many challenges in the treatment of solid tumors, including insufficient tumor invasion, an immunosuppressive microenvironment, and limited persistence of infused T cells in vivo, leading to poor efficacy and a high relapse rate.

[0003] T-cell receptor fusion constructs (TRuC) are an emerging genetically engineered T-cell platform. Unlike CAR structures that utilize only CD3ζ signaling, TruC fuses a tumor-specific single-chain antibody (scFv) with the CD3ε subunit of the natural TCR-CD3 complex, enabling it to utilize the complete TCR signaling pathway. In preclinical studies, it has shown superior solid tumor infiltration ability and safety compared to CAR-T cells.

[0004] Urokinase plasminogen activator receptor (uPAR) is a glycosylphosphatidylinositol-anchored membrane protein that is overexpressed in a variety of solid tumors (such as breast cancer, colorectal cancer, and glioma) and hematologic malignancies, while its expression level is low in normal tissues, making it a broad-spectrum and promising target for cancer therapy.

[0005] Improving the persistence, proliferation capacity, and homing / infiltration efficiency of engineered T cells in vivo is key to enhancing their therapeutic efficacy. Interleukin-7 (IL-7) is a crucial cytokine for maintaining T cell homeostasis, promoting their survival, proliferation, and memory formation. Platelet-3 factor 4 (PF4), also known as CXCL4, is a pleiotropic chemokine with immunomodulatory functions, capable of chemotactically attracting various immune cells (including T cells) and inhibiting tumor angiogenesis.

[0006] Based on this, the present invention combines the TRuC-T cell platform, uPAR target, and the immune-enhancing functions of IL-7 and PF4, aiming to develop a novel engineered T cell therapy with stronger anti-tumor activity and durability. Summary of the Invention

[0007] To address the problems and deficiencies in the aforementioned background technologies, this invention provides a genetically engineered T cell with enhanced anti-tumor activity and improved durability, specifically targeting the shortcomings of existing CAR-T or simple TruC-T cell therapies. This T cell, which targets the urokinase-type plasminogen activator receptor (uPAR) and simultaneously secretes and expresses the cytokine interleukin-7 (IL-7) and the chemokine platelet factor 4 (PF4), is described in the invention. The invention also includes its preparation method and its application in treating uPAR-positive tumors.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] This invention provides a TRuC-T cell that targets uPAR and secretes IL-7 and PF4. The pLenti-EF1α-Anti-uPAR TRuC-P2A-IL-7-T2A-PF4 plasmid vector (secreting both IL-7 and PF4) was constructed using genetic engineering technology. Then, a lentiviral vector was packaged using a high-titer, high-purity lentiviral large-scale production process and transduced into activated human CD3+ T cells. After five days of culture, the expression rate of Anti-uPAR scFv was detected by flow cytometry, and the in vitro verification showed that Anti-uPAR TRuC(PF4×IL-7)-T cells may possess more durable anti-tumor activity.

[0010] A type of TRuC-T cell that secretes and expresses IL-7 and PF4 to enhance anti-tumor activity and targets uPAR, characterized in that the TRuC-T cells are transduced and express a fusion gene construct encoding a target uPAR-targeting TRuC-T cell that enhances anti-tumor activity via a recombinant lentiviral expression vector.

[0011] Preferably, a fusion gene construct encoding uPAR-targeting TRuC-T cells that enhances antitumor activity comprises: (a) A single-chain antibody variable region fragment (scFv) that specifically targets the urokinase-type plasminogen activator receptor (uPAR); (b) The CD3ε subunit of the T cell receptor (TCR)-CD3 complex or a functional fragment thereof; (c) The coding sequence for interleukin-7 (IL-7); and / or (d) The coding sequence of platelet factor 4 (PF4); Preferably, scFv is fused with the CD3ε subunit or its functional fragment to form a TruC core structure, and the coding sequences of IL-7 and / or PF4 are linked to the TruC core structure via self-cleaving peptide linking sequences.

[0012] Preferably, the self-cleaving peptide is a P2A peptide, a T2A peptide, or a combination thereof.

[0013] Preferably, the recombinant lentiviral expression vector comprises the fusion gene construct as described in claim 2.

[0014] Preferably, TRuC-T cells constitutively secrete IL-7 and PF4.

[0015] The application of a type of TRuC-T cell that secretes and expresses IL-7 and PF4 to enhance antitumor activity targeting uPAR in the preparation of an antitumor drug composition.

[0016] Preferably, the antitumor drug composition uses TRuC-T cells that secrete and express IL-7 and PF4 and target uPAR as the active ingredient, and exhibits one or more of the following properties: (a) Enhanced in vitro proliferation capacity; (b) Enhanced chemotactic effect on T cells; (c) Maintain a higher proportion of CD8+ T cells and a higher proportion of poorly differentiated memory T cell (TSCM / TN / TCM) subsets; (d) It does not increase the expression of cell surface immune checkpoints (PD-1, TIM-3); (e) It has highly efficient and specific killing activity against uPAR-positive tumor cells.

[0017] Preferably, the cancer is a cancer that highly expresses uPAR, including one or more of breast cancer, colorectal cancer, glioma, and hematologic malignancies.

[0018] A method for preparing uPAR-targeting TRuC-T cells that secrete and express IL-7 and PF4 to enhance anti-tumor activity, comprising the following steps: S1: Construct a recombinant lentiviral expression vector comprising the fusion gene construct of claim 5; S2: Co-transfect packaging cells with the recombinant lentiviral expression vector from step S1 and the packaging plasmid to obtain recombinant lentivirus; S3: Isolate human peripheral blood mononuclear cells and activate CD3 and T cells; S4: The recombinant lentivirus obtained in step S2 is used to transduce the CD3 and T cells activated in step S3, and the TRuC-T cells are obtained after culturing.

[0019] The TRuC-T cells of the present invention, especially the Anti-uPAR TRuC(PF4×IL-7)-T cells that simultaneously secrete IL-7 and PF4, have the following beneficial effects: (1) Highly effective targeting and killing: It can specifically recognize and efficiently kill various uPAR-positive tumor cell lines (such as MDA-MB-231 breast cancer cells and U-87MG glioma cells), while having no killing effect on uPAR-negative cells.

[0020] (2) Self-proliferation and maintenance: The secreted IL-7 can significantly promote the proliferation of TRuC-T cells in an autocrine / paracrine manner, and can also maintain a stronger proliferative capacity in an in vitro simulated tumor stimulation environment, which is beneficial to the maintenance of the number of T cells in vivo.

[0021] (3) Immune cell chemotaxis and recruitment: The secreted PF4 can effectively chemotize T cells, and theoretically can recruit more endogenous or infused T cells to the tumor site, thereby enhancing the infiltration of immune cells in the tumor microenvironment.

[0022] (4) Optimize T cell subsets: The introduction of IL-7 helps maintain a high proportion of CD8+ T cells and a higher proportion of low-differentiation memory T cell subsets (such as TSCM and TN) with more stem cell characteristics and stronger proliferative potential, indicating more durable in vivo anti-tumor immunity.

[0023] (5) Good safety: The introduction of IL-7 and PF4 does not lead to an increase in the expression of inhibitory immune checkpoints (such as PD-1 and TIM-3) on the surface of TRuC-T cells. In fact, the introduction of PF4 may even partially reduce the expression of TIM-3, indicating that it does not exacerbate T cell exhaustion.

[0024] (6) Synergistic enhancement of in vivo efficacy: In the MDA-MB-231 breast cancer model of NOD-SCID mice, the Anti-uPARTRuC(PF4×IL-7)-T cell therapy group showed the strongest tumor growth inhibition effect and the least tumor residue. Its efficacy was better than that of variants of IL-7 or PF4 introduced alone and basic TRuC-T cells, which proved the synergistic anti-tumor effect of IL-7 and PF4 in vivo.

[0025] The novel TRuC-T cells of this invention, while utilizing the signal transduction and targeted killing activity of the intact TCR-CD3 complex, simultaneously introduce IL-7 cytokine and PF4 chemokine as functional enhancement modules. IL-7 can effectively promote the survival, proliferation, and long-term maintenance of T cells (especially memory T cell subsets), while PF4 can chemotactically attract and recruit various immune cells to the tumor microenvironment, synergistically enhancing the infiltration capacity of T cells and local immune responses, thereby significantly improving anti-tumor activity, prolonging the duration of action in vivo, and effectively reducing the risk of tumor recurrence. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0027] Figure 1 This is a schematic diagram of the four TRuC plasmid structures targeting uPAR and a schematic diagram of their complex with the natural TCR.

[0028] Figure 2 The expression rate of Anti-uPARscFv on the cell surface of T cells transduced with four types of TruC lentiviruses was detected by flow cytometry, proving that TruC-T cells were successfully prepared.

[0029] Figure 3 The proliferation assays using ELISA and CFSE demonstrated that Anti-uPAR TRuC(IL-7)-T and Anti-uPARTRuC(PF4×IL-7)-T cells can effectively secrete IL-7 and have stronger proliferative capacity.

[0030] Figure 4 The results, obtained through ELISA and Transwell chemotaxis assays, demonstrated that Anti-uPAR TRuC(PF4)-T and Anti-uPAR TRuC(PF4×IL-7)-T cells can effectively secrete PF4 and have a chemotactic effect on T cells.

[0031] Figure 5 The results, obtained through luciferase killing assay and CD107a degranulation detection, demonstrated that all TruC-T cells could efficiently and specifically kill uPAR-positive target cells, and that the introduction of IL-7 enhanced the killing activity.

[0032] Figure 6 Flow cytometry analysis showed that the introduction of IL-7 and PF4 after co-incubation with target cells helped maintain a high proportion of poorly differentiated memory cells (TSCM+TN+TCM) in CD8+TRuC-T cells and optimize the CD8+ / CD4+ ratio.

[0033] Figure 7 Flow cytometry analysis showed that the introduction of IL-7 and PF4 did not increase the expression of the immune checkpoints PD-1 and TIM-3 on the surface of TruC-T cells.

[0034] Figure 8In vivo experiments using a mouse breast cancer model demonstrated that Anti-uPAR TRuC(PF4×IL-7)-T cells have the strongest tumor-suppressive effect and good safety. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0036] Example Main experimental materials: Eukaryotic expression vector pLenti and packaging plasmids pLP1, pLP2, and pMD2.G (from laboratory storage), HEK-293T cell line (human embryonic kidney cells), MM.1S cell line (human multiple myeloma cells), MDA-MB-231 cell line (human breast cancer cells), and U-87 MG cell line (human glioblastoma cells), MluI-HF and EcoRI-HF restriction endonucleases (NEB), high-fidelity Prime GXL STAR enzyme (TAKARA), GeneRuler 1 kb Plus DNALadder (Thermo Fisher Scientific), TransStbl3 competent cells (TransGen Biotech), Plasmid Mini Kit I (OMEGA), EndoFree® Plasmid Maxi Kit (QIAGEN), GoldView type I nucleic acid dye (Solepro Science & Technology Co., Ltd.), and agarose (BIOWEST, Spain). The company provides DNA gel extraction kits (Axygen, USA), DMEM, RPMI-1640, Opti-MEM medium, Gibco FBS (Thermo Fisher Scientific), Sanger sequencing (Shanghai Sangon Biotech Co., Ltd.), NaCl, yeast extract, peptone, EDTA, NaOH (Shanghai Sangon Biotech Co., Ltd.), primers (Jiangsu Genewise Biotech Co., Ltd.), Anti-Fab-Bio and uPAR-Bio recombinant proteins (Beijing Biosciences Co., Ltd.), PE-Streptavidin, APC-Streptavidin, PE-anti-human PD-1 antibody, FITC-anti-human TIM-3 antibody, FITC-anti-human CCR7 antibody, APC-anti-human CD45RA antibody, PE / cy7-anti-human CD8 antibody, FITC-anti-human CD4 antibody, and PE-anti-human CD107a antibody (BioLegend, USA).

[0037] I. Construction of recombinant plasmids ① Construct the recombinant plasmid pLenti-EF1α-Anti-uPAR TRuC-P2A-IL-7-T2A-PF4: Plasmid maps and primers for Anti-uPAR TRuC, Anti-uPAR TRuC(IL-7), Anti-uPAR TRuC(PF4), and Anti-uPAR TRuC(PF4×IL-7) were designed. The Anti-uPAR TRuC and IL-7 DNA fragments were obtained from laboratory storage, and the PF4 gene sequence was acquired from the Uniprot database. The PF4 gene fragment and PCR primers were synthesized at Suzhou Genewiz Biotechnology Co., Ltd. The required DNA template fragments for plasmid construction were added to PCR tubes according to the PCR system volume, as shown in Table 1. The reagents were briefly centrifuged to the bottom of the tube. The PCR program was run in the PCR instrument to amplify the DNA fragments. After completion, the PCR products were removed and cooled. The plasmid vector and the synthesized fragments were double-digested with EcoRI-HF and MluI-HF, ensuring both had EcoRI and MluI sticky ends. The reaction conditions were 37℃ for 3 hours and 65℃ for 20 minutes. The digestion system is shown in Table 2. The enzyme digestion products were subjected to 1% agarose gel electrophoresis to obtain the vector fragment. The Plenti vector fragment and the target fragment were then recovered using the XYGENE gel recovery kit (the operation steps are shown in Table 3 below), and the concentration and purity were detected. The vector fragment and the target fragment were seamlessly cloned (system shown in Table 4), 16℃ for 16-24h, 65℃ for 10min, and then transformed into plasmids (the seamless cloned product was placed on ice for 5min, then transferred into 50ul TransStbl3 competent cells, placed on ice for 30min, 42℃ for 45s, then on ice for 5min, 500ul LB was added, and activated in a shaker at 37℃ and 250rpm / min for 1h, then centrifuged at 5000rpm / min at 20℃ for 5min, the supernatant was discarded, the remaining bacterial culture was mixed and plated, and incubated at 37℃ for 12-14h). Single colonies were selected for bacterial amplification at 37℃, 250 rpm / min, for 12-14 hours. Plasmids were extracted, and finally identified by restriction endonuclease digestion with MluI-HF and EcoRI-HF. Sanger sequencing was then performed, and after comparison, the correct plasmids were successfully extracted for subsequent applications. The structures of the four recombinant plasmids constructed are shown below. Figure 1 As shown in Figure A, the structural comparison between the TRuC fusion protein and the natural TCR complex is as follows: Figure 1 As shown in B.

[0038] Table 1 PCR system

[0039] Table 2 Restriction Enzyme Digestion System

[0040] Table 3 Adhesive Recycling

[0041] Table 4 Seamless Cloning

[0042] II. Using Plenti vector plasmid and helper plasmid to transduce 293T cells for lentivirus packaging (1) Culture 293T cells in a 15cm cell dish. When the 293T cells reach 70% of the full field of view, resuspend 60ug of PEI in 1.5ml PBS and resuspend the Plenti vector plasmid and helper plasmid with a total mass of 20ug in 1.5ml PBS. (2) Let stand at room temperature for 5 min, then add the PBS-PEI mixture to the PBS-DNA mixture and let stand at room temperature for 20 min; (3) Prepare OPTI-DMEM whole culture and warm it in a 37℃ incubator. Discard the original DMEM culture medium in the 293T cells and add OPTI-DMEM along the wall of the dish to the 293T cells. (4) Add the PEI-DNA-PBS mixture to a culture dish and incubate at 37°C for 48 hours; (5) Collect the lentivirus in the supernatant into a 50ml centrifuge tube, add 20ml of culture medium and incubate for 24h to collect the virus within 72h; (6) Centrifuge at 1500 rpm for 5 min to remove cell debris, or filter through a 0.45 μm filter with a syringe, centrifuge at 3000 × g for 12-14 h, and concentrate the virus at 4 °C. (7) Discard the supernatant and add Vivo or AIM-V whole culture at a ratio of 1:200-1:400 (preferably with 1% HEPES) to resuspend the virus; (8) Aliquot the virus into 1.5ml Ep tubes and store at -80℃, avoiding repeated freeze-thaw cycles (freeze-thaw cycles will reduce the titer by an order of magnitude).

[0043] III. Peripheral blood mononuclear cells (PBMCs) from healthy individuals were isolated by density gradient centrifugation, and activated human CD3+ T cells were transduced with lentiviruses. The expression of Anti-uPAR scFv on the surface of T cells was then detected.

[0044] (1) Take 10 ml of peripheral blood from a healthy person into an EDTA-Na2 anticoagulant tube and mix it with DPBS at a ratio of 1:1; (2) Take four 15ml sterile centrifuge tubes, add 5ml of Ficoll separation solution to each tube, and slowly add the mixture of peripheral blood and DPBS to the surface of the Ficoll separation solution, being careful not to disturb the surface of the solution; (3) Centrifuge horizontally at 800g for 20 minutes at 25℃, with both acceleration and deceleration set to "0"; (4) After centrifugation, use a Pasteur pipette to aspirate the white flocculent layer, i.e., the PBMC layer, from the centrifuge tube, place it in a new sterile centrifuge tube, add PBS, and centrifuge and wash the PBMC twice. (5) Centrifuge horizontally at 1500 rpm / min for 5 min, discard the supernatant, add 1 ml Buffer1 (DPBS containing 5% FBS) to resuspend and count PBMCs; (6) The proportion of CD3 positive cells in PBMCs was determined by flow cytometry. CD3 / CD28 beads were added to the cell suspension at a ratio of CD3 / CD28 dynabeads:CD3 positive cells = 3:1 (30 μL beads for 106 CD3 positive cells), and the mixture was rotated and shaken at 1 rpm for 30 min at 4°C to ensure that the magnetic beads and cells were in full contact and bound. (7) After 30 minutes, add enough (greater than 1 ml) Buffer 1 to the test tube, then place the test tube on a magnetic rack and rotate it left and right for 1-2 minutes, and discard the supernatant; use an appropriate amount of T to treat the cell-magnetic bead conjugate. Resuspend the cells in the culture medium, seed them into appropriate cell culture plates according to cell density, and incubate at 37°C. Activate and culture in a 5% CO2 incubator for 24-48 h.

[0045] (8) After the activated human CD3+ T cells are gently dispersed by pipetting, 10 μL is taken for counting; (9) Add culture medium to maintain the CD3 positive cell concentration at 0.5-1x10⁻⁶. 6 Spread between / ml in a 96-well plate; (10) Virus transduction: Add the lentivirus to be transduced into a 96-well plate containing T cells, add T cell culture medium to make up to a total volume of 200 μL, add 1 μL of Polybrene (which promotes lentivirus transduction to T cells) diluted 10 times with T cell culture medium to each well, centrifuge at 32°C, 1200×g for 90 min, and then place in a 37°C, 5% CO2 incubator for incubation. (11) Change medium: After 4 h, carefully remove the cell culture plate and aspirate 100 μL of supernatant, then add 120-150 μL of fresh T cell culture medium to prevent the toxic effects of Polybrene from affecting the growth status of T cells; (12) Culture: Observe T cells under a microscope daily, and adjust the medium and expand the culture plate according to cell status and density. Count cell growth on days 1, 3, and 5, and plot the growth curve. Measure the expression rate of Anti-uPAR scFv on the T cell surface at days 5-7. Results are as follows: Figure 2 As shown.

[0046] IV. Detection of the killing effect of TRuC-T cells on target cells by luciferase method, co-incubation experiment, ELISA and CFSE cell proliferation experiment.

[0047] Prepare TRuC-T cells in advance; culture target cells (MM.1S cells, luciferase-containing cells, etc.) in advance. MDA-MB-231 cells and U-87 MG cells).

[0048] 1) Target cell treatment: Digest and treat target cells from cell culture dishes, transfer to centrifuge tubes, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL of target cell culture medium and count the cells; 2) Target cell plating: Based on the required number of wells for the experiment, plate the target cells at a rate of 0.01 × 10⁶ cells / well in a 96-well plate, add an appropriate amount of culture medium, and incubate at 37°C in a 5% CO₂ incubator for 4-6 hours to allow them to adhere to the plate before proceeding with subsequent experiments. 3) T cell treatment: Transfer sufficient quantities of TruC-T cells and MOCK-T cells from each experimental group, whose TruC expression on the cell surface has been detected by flow cytometry, to centrifuge tubes, centrifuge at 1700 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL of T cell culture medium, demagnetize and count the cells; 4) Adjusting TruC expression rate in TruC-T cells of each experimental group: Based on TruC expression and counting results, the TruC expression of T cells in each experimental group with high TruC expression rate was adjusted using MOCK-T cells until it was consistent with the TruC expression of T cells in the experimental group with the lowest TruC expression rate; 5) Adding T cells: Based on different target cell experimental conditions, add TruC-T cells from each experimental group to the target cell wells at an equivalent target ratio of TruC-T cells:target cells of 1:4, 1:2, 1:1, or 2:1. The group was supplemented with the same number of MOCK-T cells as the experimental group under each effector-target ratio condition (i.e., the TruC expression rate was zero, but the total number of T cells was kept consistent with the experimental group). 6) Replenishment: Add cell culture medium to each well to 200 μL. Add ddH2O to the target cell wells of the positive control wells to 200 μL. Add target cell culture medium to the target cell wells of the negative control wells to 200 μL. Incubate at 37°C in a 5% CO2 incubator. 7) Detection of fluorescence value: After co-incubation for an appropriate time, centrifuge the cell culture plate at 2500 rpm for 3 min, discard the supernatant, add 200 μL of detection medium (D-luciferin:DMEM empty culture = 1:200) to each well, incubate at room temperature in the dark for 10 min, and then detect the bioluminescence value using an ELISA reader; 8) Calculation: Kill rate = (Fluorescence value of negative control well - Fluorescence value of detection well) / (Fluorescence value of blank negative control well - Fluorescence value of blank positive control well) × 100%. Results are as follows: Figure 4 , 5 As shown TRuC-T cell co-incubation experiment with target cells The co-incubation of TRuC-T cells with target cells is primarily to detect the effects of tumor cell activation on TRuC-T cells. The expression of various T cell surface markers and the secretion of various proteins in the supernatant were studied. The co-incubation effector-target ratio and time were specifically set according to the detection indicators. After co-incubation, different treatments were performed depending on the experimental purpose: ① The co-incubated cells were used for flow cytometry to detect the expression of cell surface markers; ② The culture supernatant was collected, centrifuged at 2500 rpm for 10 min to remove the precipitate, and the supernatant was immediately used for subsequent detection experiments or stored at -20℃ for later use. Results are as follows... Figure 3 , 5 As shown in Figures 6 and 7.

[0049] ELISA 1) Prepare 1× wash buffer and 1× detection buffer in advance, and dissolve and dilute the standard in advance; 2) Soaking the microplate: Take the required amount of microplate for the experiment, add 300 μL of 1× washing buffer and let it soak for 30 seconds. Discard the washing buffer and pat the wells dry on absorbent paper (after washing the plate, quickly proceed with subsequent operations and do not let the wells dry). 3) Add standard: Add 100 μL of the standard that has been serially diluted 2 times to the standard well, and add 100 μL of standard diluent to the blank well; 4) Add sample: Add 100 μL of the sample to be tested to the sample well (the sample can be appropriately diluted with 1× detection buffer depending on the detection range and preliminary experimental results); 5) Add detection antibody: Add 50 μL of detection antibody working solution (1× detection buffer diluted 1:100) to each well, ensuring that steps 3), 4), and 5) are added continuously and completed within 15 min; 6) Incubation: Seal the plate with a sealing film, and incubate at room temperature and 100-300 rpm using a horizontal shaker for 2 hours (ensuring that the solution in each well does not spill out and is thoroughly mixed). 7) Washing: Discard the liquid in the detection plate, add 300 μL of 1× washing solution to each well and wash the plate 6 times. After each wash, discard the supernatant and pat the plate dry on absorbent paper. 8) Enzyme incubation: Add 100 μL of streptavidin working solution (1× detection buffer diluted 1:100) to each well, seal the plate with a new sealing film, and incubate at room temperature and 100-300 rpm using a horizontal shaker for 45 min (ensuring that the solution in each well is not spilled and is thoroughly mixed). 9) Washing: Repeat step 7); 10) Add substrate for color development: Add 100 μL of TMB substrate to each well and incubate at room temperature in the dark for 5-30 min; 11) Add stop solution: Add 100 μL of stop solution to each test well, gently tap the plate frame to mix it thoroughly, and the color will change from blue to yellow; 12) Detection reading: Within 30 minutes, use an ELISA reader to perform dual-wavelength detection, measuring the OD value at the maximum absorption wavelength of 450 nm and the reference wavelength of 630 nm; 13) Calculation: Calculate the concentration of the analyte based on the standard curve and the dilution factor of the test sample. Results are as follows: Figure 3 , 4 7.

[0050] CFSE cell proliferation assay 1) Target cell treatment: Digest and treat the target cells from the cell culture dish, transfer them to a centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL of target cell culture medium and count the cells. Based on the required cell quantity for the experiment, plate the target cells into a 24-well cell culture plate and allow them to adhere for 4-6 h before proceeding with subsequent experiments. 2) T cell treatment: Transfer sufficient quantities of TruC-T cells and MOCK-T cells from each experimental group, whose TruC expression on the cell surface has been detected by flow cytometry, to centrifuge tubes, centrifuge at 1700 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL PBS to demagnetize and count the cells; 3) Adjusting the TruC expression rate of TruC-T cells in each experimental group: Based on the TruC expression and counting results, the TruC expression of T cells in the experimental group with high TruC expression rate was adjusted using MOCK-T cells until it was consistent with the TruC expression of T cells in the experimental group with the lowest TruC expression rate; 4) CFSE labeling of T cells: Take the required amount of each TruC-T group and MOCK-T group cells into Eppendorf tubes, centrifuge at 1700 rpm for 5 min, discard the supernatant, and resuspend the cell pellet in 100 μL of CFSE dilution buffer (CFSE stock solution:PBS = 1:1000). The number of cells that can be stained by 100 μL of CFSE dilution buffer is in the range of 1-10 × 10⁶. Incubate at room temperature in the dark for 15-20 min, and occasionally pipette the cells to ensure more thorough and uniform staining. 5) Quenching unbound CFSE: After the labeling time is up, add 1 mL of RPIM-1640 complete medium containing 10% FBS to the Ep tube to stop the reaction and quench the unbound CFSE at the same time. Centrifuge at 1700 rpm for 5 min and discard the supernatant. 6) Washing: Resuspend the cell pellet in 1 mL PBS, centrifuge at 1700 rpm for 5 min, and discard the supernatant; 7) Resuspend T cells from each group in T cell culture medium, and take 0.5×10⁶ TruC-T cells from each group and seed them into 24-well plates. Add culture medium to the total liquid volume to 1 mL, and then incubate at 37℃ in a 5% CO₂ incubator (for the proliferation detection experiment after target cell activation, seed the T cells into the cell culture plate with the target cells in step 1 and co-incubate with the target cells). Detect the baseline value of CFSE markers in the remaining cells using flow cytometry. 8) After 5-7 days of culture, collect cultured cells and use flow cytometry to detect the decrease in CFSE-labeled fluorescence to determine proliferation. Results are as follows: Figure 3 .

[0051] V. Chemotactic effect of Anti-uPAR TRuC(PF4×IL-7)-T cells on T cells 1) Collection of T cell supernatant from experimental groups: After T cells are activated and transduced with lentivirus, the supernatant of TruC-T cells cultured to day 5-7 is collected from each group. The cells are centrifuged at 2500 rpm for 10 min to remove cell debris. 400 μL of the culture supernatant from each group is placed in the lower chamber of a Transwell for later use. 2) MOCK-T cell treatment: Transfer MOCK-T cells to centrifuge tubes, centrifuge at 1700 rpm for 5 min, discard the supernatant, resuspend the cell pellet in 1 mL PBS to demagnetize and count the cells, centrifuge the remaining cells at 1700 rpm for 5 min, and discard the supernatant; 3) CFSE labeling of MOCK-T cells: The cell pellet was resuspended in 100 μL of CFSE dilution buffer (CFSE stock solution:PBS = 1:1000). The number of cells that can be stained by 100 μL of CFSE dilution buffer ranges from 1 to 10 × 10⁶. The cells were incubated at room temperature in the dark for 15-20 min, during which the cells were pipetted from time to time to ensure more thorough and uniform staining. 4) Quenching unbound CFSE: After the labeling time is up, add 1 mL of RPIM-1640 complete medium containing 10% FBS to the Ep tube to stop the reaction and quench the unbound CFSE at the same time. Centrifuge at 1700 rpm for 5 min and discard the supernatant. 5) Washing: Resuspend the cell pellet in 1 mL PBS, centrifuge at 1700 rpm for 5 min, and discard the supernatant; 6) After staining, the cell pellet was resuspended in T cell culture medium, counted, and the cell density was adjusted to 0.1×10⁶ / 100 μL; 7) Add 200 μL of CFSE-labeled cells to the upper chamber of a Transwell chamber and incubate at 37°C in a 5% CO2 incubator for 2-4 h; 8) Observation: After 2-4 hours, remove the upper chamber, mix the liquid in the lower chamber of the Transwell chamber, let it stand for 10 minutes, and then observe the T cells that have migrated to the lower chamber under a fluorescence microscope (CFSE-labeled cells will emit green fluorescence). Take three random field-of-view photographs under 20x magnification. Results are as follows: Figure 4 .

[0052] VI. Establishment of a mouse breast cancer model Female NOD-SCID mice aged 4-6 weeks, purchased from Vital River Pharmaceuticals in Beijing, were acclimatized in an SPF-grade animal facility for one week. MDA-MB-231 cells in good condition were washed with pre-cooled PBS and counted at 0.5 × 10⁻⁶ cells per mouse. 6 Cells were resuspended in a PBS-Matrix gel mixture and injected subcutaneously in 200 μL. Tumor formation was confirmed by IVIS imaging on days 5-7 post-injection. After tumor formation, mice were randomly divided into five groups, receiving either an equal volume of MOCK-T cells with consistent TruC expression rates or different modified TruC-T cells (including groups expressing IL-7 / PF4 alone or in combination). During treatment, tumor growth was monitored every 5 days by intraperitoneal injection of D-fluorescein combined with IVIS in vivo imaging; mouse status and body weight were also regularly observed to assess safety. Mice were sacrificed on day 30 of treatment, and tumors were dissected to prepare single-cell suspensions. The proportion of residual tumor cells within the tumor was detected by flow cytometry using GFP protein expressed by the tumor cells. Results are as follows: Figure 8 .

[0053] VII. Bioluminescence Imaging 1) Turn on the instrument and computer, and access the software. 2) Start the anesthesia machine: Check the amount of anesthetic in the canister, turn on the oxygen cylinder, adjust the oxygen flow rate to 1.5L, turn the induction anesthesia flow meter to maximum, adjust the concentration in the evaporation tank to 5%, and start the waste gas absorption device. 3) Intraperitoneally inject the substrate (200ul substrate / mouse) into the mice. After 3 minutes, place the mice in the anesthesia room for anesthesia. 4) Once the mice's breathing is stable, place them in the imaging room for imaging. Results are as follows: Figure 8 .

[0054] VIII. Data Analysis: Flow cytometry results were analyzed using Flowjo V10 software; statistical analysis and graph creation were performed using GraphPad prim 9.0 software. Experimental data are expressed as mean ± standard deviation (Mean ± SD). Comparisons between two groups were performed using t-tests, and comparisons among multiple groups were performed using analysis of variance. P < 0.05 was considered statistically significant. P < 0.05, P < 0.01, P < 0.001, P<0.0001).

[0055] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A type of TRuC-T cell that secretes and expresses IL-7 and PF4 to enhance antitumor activity and targets uPAR, characterized in that, The TRuC-T cells were transduced and expressed via a recombinant lentiviral expression vector to a fusion gene construct encoding uPAR-targeting TRuC-T cells that enhances antitumor activity.

2. The TRuC-T cells targeting uPAR that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 1, characterized in that, The aforementioned fusion gene construct encoding uPAR-targeting TRuC-T cells that enhances anti-tumor activity comprises: (a) A single-chain antibody variable region fragment (scFv) that specifically targets the urokinase-type plasminogen activator receptor (uPAR); (b) The CD3ε subunit of the T cell receptor (TCR)-CD3 complex or a functional fragment thereof; (c) The coding sequence for interleukin-7 (IL-7); and / or (d) The coding sequence of platelet factor 4 (PF4).

3. The TRuC-T cells targeting uPAR that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 2, characterized in that, The scFv is fused with the CD3ε subunit or its functional fragment to form the TruC core structure, and the coding sequences of IL-7 and / or PF4 are linked to the TruC core structure through a self-cleaving peptide linker sequence.

4. The TRuC-T cells targeting uPAR that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 3, characterized in that, The self-cleaving peptide is a P2A peptide, a T2A peptide, or a combination thereof.

5. The TRuC-T cells targeting uPAR that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 1, characterized in that, The recombinant lentiviral expression vector comprises the fusion gene construct as described in claim 2.

6. The TRuC-T cells targeting uPAR that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 1, characterized in that, The TRuC-T cells constitutively secrete IL-7 and PF4.

7. The application of a type of uPAR-targeting TRuC-T cell that secretes and expresses IL-7 and PF4 to enhance antitumor activity, characterized in that, It is used in the preparation of antitumor drug compositions.

8. The application of the uPAR-targeting TRuC-T cells that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 7, characterized in that, The antitumor drug composition uses TRuC-T cells that secrete and express IL-7 and PF4 and target uPAR as the active ingredient.

9. The application of the uPAR-targeting TRuC-T cells that secrete and express IL-7 and PF4 to enhance antitumor activity according to claim 8, characterized in that, The cancers mentioned are cancers that highly express uPAR, including one or more of the following: breast cancer, colorectal cancer, glioma, and hematologic malignancies.

10. A method for preparing uPAR-targeting TRuC-T cells that secrete and express IL-7 and PF4 to enhance antitumor activity, characterized in that, Includes the following steps: S1: Construct a recombinant lentiviral expression vector comprising the fusion gene construct of claim 5; S2: Co-transfect packaging cells with the recombinant lentiviral expression vector from step S1 and the packaging plasmid to obtain recombinant lentivirus; S3: Isolate human peripheral blood mononuclear cells and activate CD3 and T cells; S4: The recombinant lentivirus obtained in step S2 is used to transduce the CD3 and T cells activated in step S3, and the TRuC-T cells are obtained after culturing.

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