A CD19-targeting bifunctional bridging molecule and application thereof in improving CAR-T cell targeting and therapeutic effect

Abstract

The application discloses a CD19-targeting bifunctional bridging molecule and application thereof in improving CAR-T cell targeting and curative effect. In view of the bottleneck of solid tumor target deficiency and antigen heterogeneity, the bifunctional "bridging molecule" DBCO-CD19 is covalently anchored on metabolic marker tumor cells with azido groups (-N3) on the surface by metabolic sugar engineering and copper-free click chemistry, so that the tumor cell surface presents CD19 antigens. The strategy converts the heterogeneous tumor atlas into a single standard CD19 target, and breaks away from the dependence on natural antigen expression. With the aid of the bridging molecule, CD19 CAR-T cells can establish precise immune recognition with target cells, and induce the formation of stable immune synapses. Research proves that the strategy can drive CAR-T cells to efficiently remove various tumors, significantly enhance deep infiltration of three-dimensional tumor spheres, and exhibit significant and continuous tumor regression.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a CD19-targeting bifunctional bridging molecule and its application in improving the targeting and efficacy of CAR-T cells. Background Technology

[0002] Malignant tumors are among the leading threats to human health worldwide. In recent years, cell immunotherapy, represented by chimeric antigen receptor T cells (CAR-T cells), has made groundbreaking progress in the treatment of hematologic malignancies, with CD19-targeted CAR-T products already approved and widely used clinically. However, applying CAR-T therapy to solid tumors still faces significant obstacles, primarily due to the lack of highly specific and uniformly expressed tumor-associated antigens (TAAs) in solid tumors. Furthermore, "antigen loss" caused by gene mutations or epigenetic silencing, along with the severe risk of off-target toxicity, further limits treatment efficacy. Therefore, current clinical therapeutic targets remain highly limited and lack broad applicability. For example, approved therapies are mainly limited to single hematologic markers such as CD19 and BCMA, and clinical exploration for solid tumors often relies on single, highly specific antigens: such as HER2 in breast cancer or EGFRvIII in glioblastoma. These single-target strategies not only fail to cover the diverse types of tumors, but their clinical efficacy is also often severely limited by tumor heterogeneity and rapid antigen escape. In summary, developing a broad-spectrum, safe, and efficient targeting strategy independent of natural tumor surface antigens has become a key scientific challenge for expanding the clinical application of CAR-T therapy.

[0003] To overcome the limitations of tumor targeting, researchers have developed various universal CAR-T platforms, including the UniCAR system, which modulates activity through intermediate adaptor proteins, and the SUPRA CAR system, based on non-covalent interactions. While these strategies improve flexibility by separating the recognition domain from the signaling module, their adaptor proteins still inherently rely on recognizing specific native proteins on the tumor surface. The combination of bioorthogonal chemistry and metabolic glycoengineering (MGE) opens new avenues for overcoming this bottleneck. Existing research introduces chemically reactive "chemical reporter groups" (such as ketones, subsequently evolving into azides) into glycan metabolic networks, making MGE a feasible strategy for specific, programmable chemical modification of living cell surfaces. Because tumor cells have a high demand for glycolysis, exogenous non-natural sugar analogs (such as chemically modified metabolic marker sugar probes carrying azido groups: tetraacetylated N-azidoacetylgalactosamine, Ac4GalNAz) can be taken up and metabolized by cells, ultimately displaying a high density of "artificial receptors"—azido groups (-N3)—on the tumor glycocalyx. This labeling pattern based on metabolic characteristics rather than specific mutated genes provides a foundation for targeting strategies that do not depend on the expression of natural antigens.

[0004] In recent years, this "artificial target" strategy has been successfully applied to targeted nanomedicine delivery and in vivo imaging, demonstrating excellent specificity in complex physiological environments. Recently, Zhao et al. innovatively applied click chemistry to engineer CAR-T cells themselves, enhancing their invasive ability against solid tumors by modifying hyaluronidase on the surface of T cells [Zhao Y, Dong Y, Yang S, et al. Bioorthogonal Equipping CAR-T Cells with Hyaluronidase and Checkpoint Blocking Antibody for Enhanced Solid Tumor Immunotherapy[J]. ACS central science, 2022,8(5):603-614.]. Similarly, Han et al. utilized this orthogonal click chemistry mechanism to drive the targeted delivery of biomimetic photothermal agents deep into solid tumors, achieving significant tumor suppression effects [Han Y, Pan H, Li W, et al. T Cell Membrane Mimicking Nanoparticles with Bioorthogonal Targeting and Immune Recognition for Enhanced Photothermal Therapy[J]. Advanced science (Weinheim, Baden-Wurttemberg, Germany), 2019,6(15):1900251.]. However, the above studies mainly focus on the delivery system or the direct modification of the effector cells themselves. How to directly reconstruct tumor-targeting recognition function using such orthogonal chemical bonds and induce the formation of stable immune synapses between effector cells and target cells still requires further exploration. In particular, how to cleverly utilize clinically mature CAR-T cells (such as CD19 CAR-T) to redirect their powerful immune-killing ability to tumor cells lacking natural targets without any additional modification or alteration to the CAR-T cells themselves remains a pressing technical bottleneck. To bridge this technological gap, a mediation strategy capable of converting "artificial targets" on the tumor surface into classical immune recognition signals is urgently needed. This invention application is therefore proposed. Summary of the Invention

[0005] The technical problem this invention aims to solve is to overcome the limited ability of existing CAR-T cell therapies to recognize antigens in solid tumors and tumors lacking specific target antigens. This invention innovatively proposes a bifunctional bridging molecule that, by coupling metabolic markers with classical antigens, achieves universal CAR-T cell targeted recognition and killing independent of natural targets.

[0006] The first objective of this invention is to provide a dual-functional universal targeted bridging molecule, DBCO-CD19.

[0007] A second objective of this invention is to provide a method for preparing the bridging molecule DBCO-CD19.

[0008] A third objective of this invention is to provide applications for the bridging molecule DBCO-CD19.

[0009] A fourth objective of this invention is to provide a universal CAR-T cell antitumor composition or kit.

[0010] The fifth objective of this invention is to provide a universal CAR-T cell targeted recognition method for tumor cells for non-disease treatment and diagnosis purposes.

[0011] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a dual-function universal targeting bridging molecule, DBCO-CD19, which is obtained by chemically modifying the tumor antigen CD19 protein with a dibenzocyclooctylene group.

[0012] This invention combines metabolic glycoengineering with bioorthogonal chemistry to propose a plug-and-play chimeric antigen receptor T-cell (CAR-T) therapy strategy based on the bifunctional bridging molecule DBCO-CD19. This strategy utilizes a copper-free click chemistry reaction to specifically covalently anchor the chemically reactive DBCO-CD19 "molecular bridge" to the surface of metabolically labeled tumor cells expressing azide groups (-N3), thereby "artificially reconstructing" classic CD19 antigen epitopes on a wide range of tumor cells and effectively decoupling target recognition from the inherent genetic background of the tumor. In vitro experiments show that this strategy can successfully induce the formation of artificial immune synapses and mediate highly efficient tumor cell killing. It not only enables CD19 CAR-T cells to effectively clear azide-labeled (-N3) K562 leukemia cells and MDA-MB-231 breast cancer cells and promote cytokine release, but also achieves deep-penetration killing in three-dimensional (3D) tumor spheroid models. Furthermore, in a zebrafish in vivo model, in vivo imaging provided a more intuitive demonstration of the dynamic clearance process of tumor cells by CAR-T cells, with the treatment group exhibiting significant tumor regression. Overall, this strategy avoids dependence on natural antigens, providing a highly efficient, broad-spectrum, and widely applicable new approach to overcome the antigenic heterogeneity of solid tumors and the targeting bottleneck of CAR-T therapy.

[0013] The present invention provides a bridging molecule that is "dual-functional" in that it can recognize N3 on tumors and can also be recognized by CD19 antibodies on the surface of CAR-T cells; the bridging molecule is "universal" in that it can recognize any tumor cells labeled with N3.

[0014] The present invention provides a method for preparing DBCO-CD19: CD19 protein is dissolved and mixed with DBCO-PEG4-NHS at a molar ratio of 1:(30~50), and reacted at room temperature in the dark for 1~3 hours. After the reaction is completed, the unreacted DBCO-PEG4-NHS is removed by ultrafiltration and washing to obtain the bridging molecule DBCO-CD19.

[0015] Preferably, unreacted DBCO-PEG4-NHS is removed by ultrafiltration washing three times (using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, 8000 rpm, 10 min).

[0016] This invention provides the application of the aforementioned bridging molecule DBCO-CD19 in enhancing the ability of CD19 CAR-T cells to target and eliminate tumor cells.

[0017] This invention provides the application of the above-mentioned bridging molecule DBCO-CD19 in the preparation of products that enhance the anti-tumor effect of CD19 CAR-T cells.

[0018] Furthermore, the bridging molecule DBCO-CD19 enhances the anti-tumor effect of CD19 CAR-T cells by converting a highly heterogeneous tumor antigen profile into a single classical CD19 target, thereby inducing a strong immune recognition and killing between CD19 CAR-T cells and different malignant tumor cells.

[0019] This invention provides the application of the above-mentioned bridging molecule DBCO-CD19 in the preparation of broad-spectrum antitumor products that are independent of natural target antigens.

[0020] The present invention provides a universal CAR-T cell anti-tumor composition or kit containing the above-mentioned bridging molecule DBCO-CD19.

[0021] Preferably, the antitumor composition or kit further comprises the metabolic marker carbohydrate precursor Ac4GalNAz and CD19CAR-T cells.

[0022] Furthermore, the Ac4GalNAz is used to display artificial receptor azide groups (-N3) at high density on the surface of tumor cells, providing covalent binding sites for the bridging molecule.

[0023] Preferably, the concentration of the bridging molecule DBCO-CD19 is 1~50 μg / mL.

[0024] This invention provides a universal CAR-T cell targeted recognition method for tumor cells for non-disease treatment and diagnosis purposes. The method uses the bridging molecule DBCO-CD19 to anchor on the surface of metabolically labeled tumor cells expressing azide groups through a copper-free click reaction. Immune cells achieve deep infiltration of solid tumors or 3D tumor sphere models and specific clearance of target tumor cells by binding to the CD19 antigen of the bridging molecule.

[0025] Preferably, the pairing groups of the copper-free click reaction are not limited to N3 and DBCO, but are preferably N3 and DBCO; the classical tumor antigen of the bridging molecule is not limited to CD19 protein, but is preferably CD19 protein; the immune cells are selected from CAR-T cells, CAR-NK cells or CAR-M cells, and are preferably CAR-T cells.

[0026] Preferably, the tumors encompass malignant tumors that lack high specificity or uniformly express natural antigens, including but not limited to leukemia and breast cancer.

[0027] In addition, the present invention also provides a method for improving the targeted recognition of tumor cells by CAR-T cells for the purpose of non-disease treatment diagnosis. The above-mentioned bifunctional bridging molecule is coupled to the surface of metabolically labeled tumor cells expressing azide group-N3. CD19 CAR-T cells target tumor cells and exert their effects by specifically recognizing CD19.

[0028] In particular, CAR-T therapy based on CD19 is mainly used to treat hematologic malignancies of B cell origin, while the DBCO-CD19 bridging strategy provided by this invention effectively decouples the relationship between target recognition and the inherent genetic background of the tumor, and can be widely applied to solid tumors that originally lacked clear targets.

[0029] The present invention has the following beneficial effects: This invention develops a universal chimeric antigen receptor T-cell (CAR-T) targeting bifunctional bridging molecule, DBCO-CD19, based on metabolic glycoengineering (MGE) and bioorthogonal click chemistry. DBCO-CD19 anchors the CD19 antigen epitope to the tumor surface via stable covalent bonds, successfully transforming the complex targeting challenges of solid tumors into a clinically mature CD19 recognition paradigm. This invention enables CAR-T cells to specifically recognize and kill target cells (N3-K562 and N3-MDA-MB-231) through DBCO-CD19. Furthermore, it significantly enhances the deep infiltration of CAR-T cells into three-dimensional (3D) tumor spheroids and mediates significant tumor regression in a zebrafish xenograft model.

[0030] The DBCO-CD19 method provided by this invention does not rely on the recognition mechanism of specific natural tumor antigen expression and can broadly bind to tumor cell membranes without depending on any specific tumor surface proteins. Experiments have demonstrated that regardless of the original surface markers of tumor cells, DBCO-CD19 can construct biologically functional CD19 antigens in cancer cell lines through a highly efficient click chemistry reaction with -N3. These findings establish the reliability of DBCO-CD19 combined with metabolic sugar engineering as a broad-spectrum and highly versatile tumor marker method. Attached Figure Description

[0031] Figure 1 The preparation method and characterization results of DBCO-CD19 molecular bridges are shown in the figure (A is the synthetic route of DBCO-CD19; B is a representative transmission electron microscope (TEM) image of unmodified CD19 protein and DBCO-CD19, with white circles indicating dispersed protein particles; scale bar: left image 50 nm, right image 20 nm; C and D are dynamic light scattering (DLS) analyses showing the hydrodynamic size distribution of CD19 and DBCO-CD19 and the corresponding statistical analysis, n=3; E is the Zeta potential analysis of CD19 and DBCO-CD19, n=3).

[0032] Figure 2 Mass spectrometry analysis results for DBCO modification (A in the figure is the major modification site Lys150; B is the major modification site Lys220; C is the major modification site Lys258).

[0033] Figure 3 The following figures illustrate the evaluation results of Ac4GalNAz metabolic labeling efficiency and stability in different tumor cell lines (Figure A shows a representative confocal microscopy image of Ac4GalNAz-labeled K562 cells, scale bar: 100 μm; B shows the representative quantitative flow cytometry analysis results of K562 cell labeling efficiency; C shows a representative confocal microscopy image of Ac4GalNAz-labeled MDA-MB-231 cells, scale bar: 100 μm; D shows the representative quantitative flow cytometry analysis results of MDA-MB-231 cell labeling efficiency; E shows the flow cytometry graph of K562 cell metabolic labeling; F shows the quantitative statistical graph of K562 cell metabolic labeling, data are expressed as mean ± standard deviation, n=3; G shows the flow cytometry graph of MDA-MB-231 cell metabolic labeling; H shows the quantitative statistical graph of MDA-MB-231 cell metabolic labeling, n=3).

[0034] Figure 4 The results of the assessment of the transduction efficiency of CD19 CAR-T cells and the binding capacity of bifunctional bridging molecules are shown in Figure A (the left panel of A shows the flow cytometry results of CD3+ T cells derived from peripheral blood mononuclear cells (PBMCs), the right panel of A shows the percentage of CD19 CAR-T cells (CAR+); B shows the flow cytometry analysis results; C and D show the results of the determination of the binding capacity of DBCO-CD19 to N3-K562 and N3-MDA-MB-231).

[0035] Figure 5 Results of DBCO-CD19 working concentration optimization (A: concentration-dependent killing effect of CD19 CAR-T cells mediated by different concentrations of DBCO-CD19 on N3-K562 and N3-MDA-MB-231 cells, n=5; B: comparison of cytotoxicity between Ac4GalNAz-labeled (N3-tumor cells, experimental group) and unlabeled tumor cells (control group), n=5).

[0036] Figure 6The in vitro targeting and cytotoxicity results of CD19 CAR-T cells against N3-K562 cells are shown in the figure (A is a schematic diagram of targeted killing; B is a confocal microscopy image, with effector cells labeled green (CAR-T / T) and target cells labeled red (N3-K562), and yellow signals indicating cell-cell contact and aggregation; scale bar: 50 μm; C and D are the flow cytometry assessment and cytotoxicity results of N3-K562 cells, n=3; E is the lysis activity of N3-K562 cells, n=3; F is interferon-γ (IFN-γ); G is tumor necrosis factor-α (TNF-α); H is interleukin-2 (IL-2), n=5; data are expressed as mean ± standard deviation, *** P <0.001, **** P <0.0001).

[0037] Figure 7 The in vitro target recognition and cytotoxicity results of CD19 CAR-T cells against N3-MDA-MB-231 cells are shown in the figure (A is a confocal microscopy image of CD19 CAR-T cells or T cells co-cultured with N3-MDA-MB-231 cells, green indicates effector cells, red indicates target cells, and yellow overlap indicates cell-cell contact; scale bar: 50 μm; B and C are the flow cytometry analysis and quantitative results of N3-MDA-MB-231 cell cytotoxicity, n=3; D is the specific lysis result of N3-MDA-MB-231 cells, n=3; E is interferon-γ (IFN-γ); F is tumor necrosis factor-α (TNF-α); G is interleukin-2 (IL-2), n=5; data are expressed as mean ± standard deviation, ** P <0.01, **** P <0.0001).

[0038] Figure 8 The results of CAR-T cell migration, invasion, and cytotoxicity in N3-K562 three-dimensional tumor spheroids are shown in the figure (A shows Transwell migration results; B shows the statistical results of the number of migrated CAT T / T cells, n=3; C shows the confocal fluorescence image of effector cells (green), and red represents the three-dimensional tumor cell spheroids, scale bar: 400 μm; D and E show the invasion ability and statistical quantitative results of CD19 CAR-T / T cells, n=3; F and G show the apoptosis / necrosis of N3-K562 cells in the three-dimensional spheroids mediated by effector cells and the corresponding statistical analysis results, n=3; data are expressed as mean ± standard deviation; ns, no statistically significant difference; **** P <0.0001).

[0039] Figure 9The results show the infiltration and cytotoxicity of CAR-T cells in N3-MDA-MB-231 three-dimensional tumor spheroids (A in the figure shows Transwell migration results; B shows the statistical analysis results of the number of migrating CAR-T T / T cells, n=3; C shows representative confocal microscopy images, scale bar: 400 μm; D and E show the migration of effector cells into N3...). - Infiltration of MDA-MB-231 three-dimensional tumor spheres and corresponding quantitative analysis results, n=3; F and G represent the cytotoxic effects and quantitative apoptosis / necrosis results of N3-MDA-MB-231 cells within the three-dimensional spheres, n=3; data are expressed as mean ± standard deviation; ns, no statistically significant difference; **** P <0.0001).

[0040] Figure 10 The following figures represent the in vivo antitumor efficacy evaluation results of a universal CAR-T targeting strategy in a zebrafish xenograft model (Figure A shows the in vivo antitumor efficacy timeline; B and D are local fluorescence microscopy images of the zebrafish tail on day 1 post-treatment, scale bar: 200 μm; C and E are longitudinal quantitative results of residual N3-K562 and N3-MDA-MB-231 tumor cells in the zebrafish tail from day 1 to day 3 post-treatment, n=5; F is a continuous time-lapse in vivo confocal imaging tracking image, with yellow arrows indicating ongoing immune killing events, scale bar: 50 μm; G is a time-lapse confocal image of targeted clearance of N3-MDA-MB-231 tumor cells, scale bar: 50 μm; data are expressed as mean ± standard deviation, **). P <0.01, *** P <0.001).

[0041] Figure 11 This is a schematic diagram of a universal CAR-T cell therapy strategy. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0043] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0044] The human K562-mCherry and MDA-MB-231-mCherry cell lines used in the examples were purchased from the China National Biomedical Experimental Cell Resource Bank; RAJI-mCherry cells were constructed by our research group. The basal culture medium (RPMI-1640 and AIM V) and additives (FBS and penicillin-streptomycin) were purchased from Gibco. Lymphoprep TM The CD19 CAR retrovirus was custom-prepared by Zhejiang Kangboyu Biotechnology Co., Ltd., and the transduction enhancer RetroNectin was purchased from Takara Bio. Human IL-2 Recombinant Protein, PeproTech®, and Dynabeads® Human T activator CD3 / CD28 for CAR-T / T cell expansion were purchased from Thermo Fisher. Ac4GalNAz was purchased from Click Chemistry Tools. Reagents used for the synthesis of DBCO-CD19: CD19 was purchased from ABclonal, and DBCO-PEG4-NHS was purchased from CONFLUORE. Reagents used for flow cytometry included FITC MouseAnti-Human CD3 (BD ​​Biosciences), FITC anti-human CD19 Antibody (Biolegend), PE anti-human EGFR Antibody (Biolegend), DBCO-Fluor 488 (Click Chemistry Tools), AFDye 488 Azide (Click Chemistry Tools), Annexin V-APC / DAPI Apoptosis Kit (Elabscience), Annexin V-FITC / DAPI Apoptosis Kit (Elabscience), Human IFN gamma Uncoated ELISA, Human TNF alpha Uncoated ELISA, Human IL-2 Uncoated ELISA, and Accumax-cell Aggregate Dissociation Medium, all purchased from Invitrogen. Hoechst 33258 and Enhanced Cell Counting Kit-8 were purchased from Beyotime. The LDH detection kit was purchased from Dojindo.

[0045] Statistical analyses of the results in the following examples were performed using GraphPad Prism software. Quantitative data are expressed as mean ± standard deviation (SD). For comparisons involving two independent groups under a single condition, Student's t-test was used; for experiments involving three or more groups under a single condition, one-way ANOVA was used, followed by Dunnett's post-hoc test for multiple comparisons of significant differences between the control and experimental groups; for experiments involving two factors, two-way ANOVA was used, followed by Dunnett's post-hoc test for multiple comparisons of significant differences between the control and experimental groups; for in vivo tumor quantification analysis longitudinally tracking the same zebrafish individual, two-way repeated measures ANOVA combined with Sidak's post-hoc test was used. P A value <0.05 was considered statistically significant.

[0046] Example 1: Preparation and characterization of DBCO-CD19 1. Preparation of DBCO-CD19 The lyophilized CD19 protein was dissolved in PBS, and the final concentration was adjusted to 0.5 mg / mL. DBCO-PEG4-NHS and CD19 were then mixed at a molar ratio of 40:1 and reacted at room temperature in the dark for 1 hour. After the reaction, unreacted DBCO-PEG4-NHS was removed by ultrafiltration (using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, 8000 rpm, 10 min) three times to obtain the DBCO-CD19 solution. The product was aliquoted and stored at -80 °C for later use. The preparation strategy of DBCO-CD19 is as follows: Figure 1 As shown in Figure A.

[0047] 2. Characterization of DBCO-CD19 Subsequently, Beijing Zhongke Baize Technology Service Co., Ltd. was commissioned to observe the morphology and dispersibility of DBCO-CD19 and CD19 using transmission electron microscopy (TEM). Particle size distribution and zeta potential were measured using a Zetasizer Nano ZS90 to assess changes in particle size and surface charge before and after modification. Then, Spectro-Zhonghe (Wuhan) Life Science Technology Co., Ltd. was commissioned to identify the specific modification sites of DBCO-PEG4-NHS on the CD19 protein using mass spectrometry.

[0048] Transmission electron microscope images such as Figure 1Figure B in the diagram shows that the prepared DBCO-CD19 consists of protein particles with uniform morphology, good dispersion, and no obvious aggregation. Dynamic light scattering (DLS) analysis further characterized the hydrodynamic particle size distribution of CD19 before and after modification, as shown in Figure B. Figure 1 As shown in Figure C. Statistical analysis showed that the hydrodynamic particle size of CD19 protein remained stably within the range of 8-9 nm before and after modification, with no statistically significant difference. P >0.05), and the particle size uniformity is good, proving that the chemical modification did not induce significant protein aggregation, such as Figure 1 As shown in D in the figure. The results of the Zeta potential analysis are as follows. Figure 1 As shown in Figure E, the surface charge shifted slightly from approximately -18 mV before modification to approximately -16 mV after modification, which preliminarily verifies the success of chemical coupling.

[0049] DBCO-CD19 was successfully synthesized by reacting the NHS ester group in DBCO-PEG4-NHS with the amino group on the side chain of the lysine residue of the CD19 protein. To definitively confirm the successful coupling of DBCO with the CD19 protein and identify the specific modification site, mass spectrometry analysis was performed on DBCO-CD19. The results are as follows: Figure 2 As shown, the modification was clearly verified, and Lys150 on the CD19 protein was precisely identified. Figure 2 Figure A in the diagram), Lys220 ( Figure 2 Figure B in the diagram) and Lys258 ( Figure 2 (Figure C) The three lysine residues are the main DBCO coupling sites.

[0050] Example 2: Tumor cell surface labeling and stability assessment based on metabolic sugar engineering technology A key prerequisite for DBCO-CD19 to serve as a bridging molecule is the introduction of the artificial target N3 onto the surface of tumor cells through metabolic glycoengineering. Considering the potential differences in metabolic activity among different tumor cell types, this example evaluated the labeling performance of Ac4GalNAz in K562 and MDA-MB-231 cells, respectively.

[0051] 1. Incorporation of non-natural sugars and bio-orthogonal markers K562 and MDA-MB-231 cells were incubated with different concentrations (0, 25, 50, 75, and 100 μM) of Ac4GalNAz for 2 days to obtain N3-K562 and N3-MDA-MB-231 cells, respectively. The presence of azide groups (-N3) on the cell surface was detected using the DBCO-Fluor 488 fluorescent probe.

[0052] The results are as follows Figure 3As shown, K562 cells were co-incubated with different concentrations of Ac4GalNAz. Confocal microscopy revealed that the fluorescence intensity corresponding to the azide group (-N3) on the cell surface increased with increasing concentration. Figure 3 (See Figure A in the figure). Flow cytometry results further confirmed that, compared with the untreated control group (50 μM), all treatment groups (25-100 μM) exhibited a significant rightward shift of fluorescence peaks, indicating that highly efficient azidation modification was successfully achieved on the cell surface. Similarly, in the solid tumor cell line MDA-MB-231, we observed significant and dose-dependent metabolic labeling effects (…). Figure 3 Figure C in the middle Figure 3 (See Figure D in the diagram). Based on the fact that the -N3 positive labeling rate on the surface of K562 and MDA-MB-231 cells both exceeded 90% at a working concentration of 50 μM, 50 μM Ac4GalNAz was selected as the working concentration for subsequent experiments.

[0053] 2. Stability and labeling efficiency determination Subsequently, target cells were pre-labeled using 50 μM Ac4GalNAz for 2 days. N3-K562 and N3-MDA-MB-231 cells, after being collected and cultured for days 0-6, were incubated with DBCO-Fluor 488 probe in the dark for 30 minutes. The membrane expression stability and labeling efficiency of the azide group were then quantitatively analyzed by flow cytometry.

[0054] The results are as follows Figure 3 As shown in the EH plot, K562 cells achieved a positivity rate of 97% on day 1, maintained at 96% on day 3, and remained at a high level of over 85% until day 6; MDA-MB-231 cells achieved a positivity rate of 99% on day 1, maintained at 99% on day 3, and still exhibited a high labeling rate of 95% until day 6. This indicates that the metabolic labeling strategy has good stability and can meet the time requirements of subsequent treatment.

[0055] Example 3: Functional verification and optimal working concentration selection of DBCO-CD19 1. DBCO-CD19 Function Verification CAR-T cell preparation: Human peripheral blood mononuclear cells (PBMCs) were isolated from the peripheral blood of healthy volunteers (18-35 years old) with normal blood routine examinations using density gradient centrifugation. Informed consent was obtained from all participants. PBMCs were seeded into AIM-V medium containing 2% heat-inactivated FBS and a final concentration of 300 U / mL recombinant human IL-2. Dynabeads were added at a 1:1 ratio of magnetic beads to cells. TMHuman T-Activator CD3 / CD28 was stimulated and amplified. Magnetic beads were removed using a magnetic rack after 48 hours. Subsequently, CD19 CAR retroviruses (5 × 10⁻⁶) were distributed according to the multiple of infection (MOI = 1). 5 Add TU (units per well) to a 24-well plate pre-coated with RetroNectin, and centrifuge at 2000 g for 2 hours at room temperature to pre-assemble the virus. After aspirating the supernatant, add 5 × 10⁻⁶ ppm of the pre-coated TU to each well. 5 Activated primary T cells were cultured and supplemented with IL-2 to a final concentration of 300 U / mL, followed by centrifugation (1000 g, 2 min). Two days after infection, the transduction efficiency of CAR-T cells was detected by flow cytometry. The results are as follows: Figure 4 Figure A in the diagram confirms the successful generation of CD19 CAR-T cells. CD3... + T cells account for 79.7% of the total number of cells (peripheral blood mononuclear cells, PBMCs), while CD3 cells account for a disproportionately large percentage. + CD19 CAR-T cells (CARs) successfully transduced in the cell population + The proportion was 65.2%.

[0056] To verify the natural antigen activity of CD19 in DBCO-CD19, DBCO-CD19 and CD19 CAR-T cells were co-incubated for 2 hours and washed twice. The binding efficiency of DBCO-CD19 to CD19 CAR-T cells was detected by flow cytometry using the AFDye 488 Azide probe. Results are as follows: Figure 4 As shown in Figure B, flow cytometry analysis confirmed that DBCO-CD19 exhibits concentration-dependent binding ability. Within the concentration range of 0 to 50 μg / mL, the higher the concentration of DBCO-CD19, the stronger the fluorescence signal detected by flow cytometry. Furthermore, the chemical modification of DBCO did not impair the natural antigenic activity of the CD19 protein; the modified molecule could still be efficiently recognized and bound by CAR-T cells.

[0057] In addition, flow cytometry analysis was performed to evaluate the binding ability of the DBCO-CD19 molecular bridge to targeted tumor cells. N3-MDA-MB-231 and N3-K562 cells pretreated with Ac4GalNAz were collected and resuspended in culture medium, followed by the addition of DBCO-CD19 to final concentrations of 0, 10, 25, and 50 μg / mL, respectively. After co-incubation at 37°C for 2 hours, the cells were thoroughly washed with PBS to remove unbound DBCO-CD19 molecules. Subsequently, the cells were stained with FITC-labeled CD19 antibody, and the corresponding FITC fluorescence signal was detected by flow cytometry to quantitatively analyze the proportion of CD19-positive cells on the surface. The concentration-dependent binding efficiency of DBCO-CD19 on the target cell surface was determined using the obtained data. Results are as follows: Figure 4 Figure C in the middle Figure 4 As shown in Figure D, at a working concentration of 50 μg / mL, the reconstruction efficiency of CD19 artificial target sites on the surface of both cell lines exceeded 95%.

[0058] 2. Optimization of DBCO-CD19 working concentration based on cytotoxicity. Based on the experimental foundation that the reconstruction efficiency of the artificial CD19 target on the target cell surface exceeded 95% at a working concentration of 50 μg / mL, its optimal killing efficacy was further optimized and confirmed through cytotoxicity assays. N3-K562 or N3-MDA-MB-231 target cells were pre-incubated with gradient concentrations (0, 10, 25, 50 μg / mL) of DBCO-CD19 for 2 hours, followed by co-culturing with CD19 CAR-T cells (effectivity-to-target ratio E:T = 5:1) for 48 hours and CCK-8 assay was performed.

[0059] The results showed that with the increase of DBCO-CD19 working concentration, its mediated immune killing activity exhibited a significant dose-dependent enhancement. Figure 5 (Figure A in the figure). Compared with the blank control group without bridging molecules (0 μg / mL, cell viability normalized to 1.0), 50 μg / mL DBCO-CD19 mediated the most potent targeted lysis, significantly reducing the proliferation and viability of N3-K562 and N3-MDA-MB-231 cells by approximately 60%. P <0.001); Notably, there was no significant difference in cytotoxic efficacy between the 25 μg / mL and 50 μg / mL groups. Considering both target remodeling efficiency and efficacy, 50 μg / mL was established as the standard working concentration for all subsequent functional experiments.

[0060] To further elucidate the killing mechanism and rule out non-specific toxicity of DBCO-CD19, parallel controls were used ( Figure 5As shown in Figure B, under the conditions of adding DBCO-CD19 (50 μg / mL) and CD19 CAR-T cells, the survival rate of N3-tumor cells was significantly reduced by about 60% compared with unlabeled tumor cells. This result confirms that DBCO-CD19 itself has no direct cytotoxicity to target cells.

[0061] Example 4: In vitro targeted recognition and cytotoxicity of CAR-T cells against hematologic malignancies and solid tumor cells. 1. Targeted analysis According to the groups, target cells labeled with Ac4GalNAz were pre-incubated with 0 / 50 μg / mL DBCO-CD19 for 2 h to covalently reconstruct the CD19 surface antigen. Then, CD19 CAR-T cells or T cells were added at an effector-to-target ratio of 5:1. After co-culturing for 6 hours, the spatial interactions and aggregation between effector and target cells were observed using confocal laser scanning microscopy.

[0062] Based on the optimized parameters and successful metabolic markers of target cells, the study evaluated whether DBCO-CD19 could act as a "molecular bridge" to mediate the recognition and killing of suspended N3-K562 cells and adherent N3-MDA-MB-231 solid tumor cells by CD19 CAR-T cells. Figure 6 As shown in Figure A, confocal microscopy imaging revealed that after 6 hours of incubation, only in the "CAR-T + DBCO-CD19" combined treatment group, green fluorescently labeled CAR-T cells significantly aggregated around red N3-tumor cells (K562 and MDA-MB-231), forming numerous green / red fluorescent co-localization signals and cell clusters. This confirmed the establishment of a tight specific binding and immune cross-linking between effector cells and target cells. Figure 6 Figure B in the middle Figure 7 (Figure A in the figure); while in each control group, the cells were randomly and discretely distributed, and no obvious specific cell aggregation was observed.

[0063] 2. Cytotoxicity test The tumor-killing efficiency after 48 hours of co-culture was quantitatively analyzed using Annexin V-FITC / DAPI double staining combined with flow cytometry. Results showed that, in a DBCO-CD19-dependent manner, the "CAR-T + DBCO-CD19" combination therapy induced significant specific tumor cell apoptosis and necrosis: in the N3-K562 hematologic malignancy model, the final apoptosis and necrosis rate of target cells was as high as approximately 60%. Figure 6 Figure C in the middle Figure 6(Figure D in the diagram), while the non-specific cell death rate in both the CAR-T group without bridging molecules and the T group with bridging molecules was less than 20%. In the highly heterogeneous N3-MDA-MB-231 solid tumor model, the combined treatment group also mediated apoptosis and necrosis rate of 63% (Figure D in the diagram). Figure 7 China B map Figure 7 (Figure C in the diagram) also showed a significant advantage in targeted clearance compared to the control group.

[0064] Acute cytotoxicity was further quantified using a 4-hour lactate dehydrogenase (LDH) release assay. LDH activity assays showed an extremely strong effect-to-target ratio dependence: at a maximum effect-to-target ratio of 10:1, the peak specific lysis rate of N3-K562 cells in the "CAR-T + DBCO-CD19" combination therapy group reached approximately 58%, which was about four times that of the CAR-T-only group (approximately 15%). Figure 6 (Figure E in the figure); the specific lysis rate of adherent N3-MDA-MB-231 solid tumor cells was as high as about 70%, which was about 2.5 times higher than that of the CAR-T group alone (only about 28%). Figure 7 (D diagram in the image).

[0065] 3. Cytokine release analysis N3-labeled tumor cells (K562 or MDA-MB-231) treated with DBCO-CD19 (+ / -) were mixed with CD19 CAR-T cells at a 1:1 ratio and co-cultured overnight. The culture supernatant was collected, and the concentrations of secreted IFN-γ, IL-2, and TNF-α were quantitatively detected using an ELISA kit according to the manufacturer's instructions.

[0066] ELISA results showed that after DBCO-CD19 specifically recognized and bound target cells, the secretion levels of key cytokines interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and interleukin-2 (IL-2) in the supernatant of CAR-T cells were significantly increased. K562 results were as follows... Figure 6 As shown in the FH diagram, the MDA-MB-231 results are as follows: Figure 7 The EG diagram is shown in the figure.

[0067] In summary, these findings demonstrate that DBCO-CD19 successfully endows CD19 CAR-T cells with novel capabilities, effectively activating CAR-T cells and triggering potent immunocytotoxicity against CD19-negative hematologic malignancies and solid tumors.

[0068] Example 5: Infiltration and cytotoxic efficacy of CAR-T cells in a three-dimensional tumor spheroid model To more realistically simulate the complex tumor microenvironment in vivo, we constructed three-dimensional (3D) tumor spheroid models of K562 and MDA-MB-231 cells.

[0069] 1. CAR-T cell migration experiment Migration experiments were performed using a Transwell chamber system with a pore size of 5.0 μm. 1×10⁻⁶ m³ of serum-free AIM-V medium was added to the upper chamber. 6 5 × 10⁵ CD19 CAR-T cells or T cells were added to the lower chamber, resuspended in serum-free AIM-V medium. 5 N3-K562 or N3-MDA-MB-231 target cells were selected, with or without DBCO-CD19 addition. After chemotactic co-culture at 37 °C for 6 hours, cells that migrated to the lower chamber were collected. Staining was performed using FITC anti-human CD3 antibody, and absolute CAR T / T cell counts were analyzed by flow cytometry.

[0070] Transwell migration experiment results are as follows: Figure 8 Figure A in the middle Figure 8 Figure B in the middle and Figure 9 Figure A in the middle Figure 9 Figure B shows that, under the specific recruitment effect of DBCO-CD19, the directional migration rate of CD19 CAR-T cells to N3-K562 cells reached 37%, while the directional migration rate to N3-MDA-MB-231 cells was as high as 48%, which showed a significant specific directional migration ability compared with the control group (the migration rate was only about 10%~20%).

[0071] 2. 3D Tumor Sphere Model Target cells pretreated with Ac4GalNAz were then used at 0.5 × 10⁻⁶. 4 Cells were seeded at a density of 100 cells / well in 96-well ultra-low adhesion V-bottom plates, followed by centrifugation at 180 g for 2 min to promote cell aggregation. Cells were cultured in RPMI 1640 medium for 3 days to form dense 3D tumor cell spheroids. After incubation with DBCO-CD19 (+ / -) for 3 hours, CAR-T cells or control T cells (5 × 10⁻⁶ cells / well) were added according to the group. 4Tumor spheres were cultured overnight (cells / well) and the cells were 1,000 per well. After culture, the tumor spheres were washed with PBS to remove loosely bound cells from the surface. The tumor spheres were stained with FITC Mouse Anti-Human CD3 and washed again, followed by confocal microscopy imaging to assess the infiltration of target cells into the deeper layers of the tumor spheres. For quantitative analysis, the tumor spheres were digested into single-cell suspensions using Accumax dissociation solution, stained with apoptosis dyes, and the proportion of CAR-T / T cell infiltration and the proportion of apoptotic target cells within the tumor spheres were analyzed by flow cytometry. Confocal microscopy further confirmed that, using DBCO-CD19 as a "molecular bridge," CD19 CAR-T cells (green) were able to overcome physical barriers and deeply infiltrate N3-K562 3D tumor spheroids. Figure 8 (Figure C in the image) Deep infiltration was also observed in the 3D tumor sphere of MDA-MB-231. Figure 9 (Figure C in the diagram).

[0072] Quantitative flow cytometry analysis confirmed that in the "CAR-T + DBCO-CD19" combination therapy group, the proportion of CAR-T cells infiltrating into the tumor spheroids surged to approximately 50% (approximately 48% in N3-K562 spheroids and approximately 51% in N3-MDA-MB-231 spheroids). This deep infiltration efficacy was highly equivalent to that of the positive control group CAR-T (RAJI) spheroids with naturally high CD19 expression (approximately 53% infiltration rate). Conversely, in the negative control groups, CAR-T / T cells were only scattered and adhered to the outermost layer of the spheroids, with a lower internal infiltration rate of only 10%–15%. Figure 8 The D diagram in the middle Figure 8 E diagram and Figure 9 The D diagram in the middle Figure 9 (Figure E in the diagram). For N3-K562 ( Figure 8 F-graph in Figure 8 (G diagram in the image) and N3-MDA-MB-231 ( Figure 9 F-graph in Figure 9 The cytotoxicity of the tumor spheroids (Figure G) was significantly higher than that of the control group. These results further demonstrate that DBCO-CD19 not only mediates contact between planar cells, but also effectively drives the deep infiltration of CAR-T cells and exerts a powerful killing effect in the complex 3D microenvironment.

[0073] Example 6: In vivo antitumor efficacy in a zebrafish tumor xenograft model Zebrafish embryos were collected after fertilization and cultured at 33°C until microinjection. On day 2 post-fertilization (2 dpf), the embryos were artificially dechorioned, anesthetized with Tricaine (200 mg / L), and fixed on slides with low-melting-point agarose. N3-labeled tumor cells and FITC-labeled effector cells (CAR-T cells) were washed repeatedly with PBS, and cell aggregates were removed using a 40 μm mesh cap. The cells were then resuspended in 2% polyvinylpyrrolidone to maintain cell dispersion stability. To facilitate in vivo high-resolution fluorescence imaging for dynamic tracking of effector cell lysis events, tumor cells and CAR-T cells were injected in a 1:1 ratio, with DBCO-CD19 added to the experimental group. The final cell concentration was adjusted to 7 × 10⁶ cells / year. 7 Cells / mL. Cell suspension was microinjected into each embryo's Cuvier tube using glass microcapillaries connected to a PLI-100A Basic Pico-Injector (Harvard Instruments). Approximately 4 nL of cell suspension was injected into each embryo. Following injection, embryos were transferred to E3 medium containing penicillin-streptomycin (1:100) and cultured at 33°C. On days 1, 2, and 3 post-injection, longitudinal fluorescence imaging was performed using in vivo confocal microscopy to quantitatively count residual tumor cells in the tail region. Time-lapse imaging was used to dynamically monitor tumor cell clearance in vivo.

[0074] Since tumor cells are not fully dispersed in the body at the initial stage of microinjection, this study selected to collect representative images starting on day 1 after treatment. Figure 10 Figure A in the diagram shows that in vivo fluorescence imaging clearly revealed that N3-K562 and N3-MDA-MB-231 tumor cells had successfully colonized the zebrafish tail region; notably, the "CAR-T + DBCO-CD19" combined treatment group showed initial efficacy one day after treatment at this time. Figure 10 Figure B in the middle Figure 10 (See Figure D in the diagram). Further longitudinal quantitative analysis of residual tumor cells in the tail from day 1 to day 3 post-treatment revealed that the CAR-T+DBCO-CD19 treatment group exhibited a significant and sustained tumor regression trend. In the experimental group, the number of tumor cells in the tail of both tumor models had decreased to less than 10 by day 3, while in the control group lacking bridging molecules, the tumor burden in zebrafish still showed a high residual tumor cell count of 20-30 by day 3. Figure 10 Figure C in the middle Figure 10 (Figure E in the diagram).

[0075] Furthermore, continuous in vivo confocal time-lapse imaging provides intuitive, dynamic visual evidence of the potent cytotoxicity of this strategy in vivo. High-resolution images clearly capture the entire process of green fluorescently labeled CAR-T cells closely contacting and specifically eliminating red fluorescently labeled N3-K562 tumor cells under hemodynamic conditions, with yellow arrows clearly indicating ongoing immune killing events. Figure 10 (Figure F in the diagram). Clearance behavior was also observed in the N3-MDA-MB-231 solid tumor model. Figure 10 (G diagram in the figure). The above in vivo data strongly support the clinical translational potential of this biotransformation-based CAR-T strategy for efficiently clearing tumors in complex biological systems.

[0076] Example 7: A CD19 CAR-T cell therapy-type targeted product This embodiment provides a universal targeted product for CD19 CAR-T cell therapy, which contains the bridging molecule DBCO-CD19, the metabolic marker carbohydrate probe Ac4GalNAz, and CD19 CAR-T cells.

[0077] First, tumor cells are labeled with the metabolically labeled carbohydrate probe Ac4GalNAz. Then, these N3-labeled tumor cells are treated with DBCO-CD19, followed by CD19 CAR-T cell therapy, thus overcoming the antigenic heterogeneity of solid tumors. Specifically, this strategy uses a copper-free click chemistry reaction to specifically covalently anchor the chemically reactive DBCO-CD19 "molecular bridge" to the surface of metabolically labeled tumor cells expressing azide groups (-N3), thereby "artificially reconstructing" the classic CD19 antigenic epitope on the tumor cells. This allows CAR-T cells to specifically recognize and kill target cells. This chemically anchored mode enables DBCO-CD19 to broadly bind to the tumor cell membrane without relying on any specific tumor surface protein. This strategy has demonstrated excellent tumor clearance and deep invasion capabilities both in vitro and in vivo, providing a feasible pathway for introducing mature CD19 CAR-T cell therapy into the treatment of solid tumors and opening new avenues for next-generation broad-spectrum tumor immunotherapy.

[0078] A schematic diagram of a universal CAR-T cell therapy strategy based on metabolic glucose engineering and bioorthogonal chemistry is shown below. Figure 11As shown, the process includes the following steps: (1) Chemical synthesis of the bifunctional bridging molecule. DBCO-CD19 was successfully synthesized by amidation of the NHS ester group in DBCO-PEG4-NHS with the side chain amino group of the lysine residue on the CD19 protein. (2) Tumor cell metabolism, click coupling and redirected immune killing. Tumor cells take up and metabolize Ac4GalNAz, causing the cell surface to express azide groups (-N3). Click chemical coupling: DBCO-modified CD19 (DBCO-CD19) acts as a "molecular bridge" and specifically binds to the -N3 group on the cell surface through copper-free click chemical coupling, causing the tumor cell surface to present CD19 antigen. CAR-T cells targeting CD19 specifically recognize the CD19 domain on the molecular bridge, thereby being activated and exerting secondary anti-tumor cytotoxicity. (3) The zebrafish xenograft model supports the clinical translational potential of this biotransformation application-oriented CAR-T strategy to efficiently clear tumors in complex biological systems.

[0079] In summary, this invention develops a universal CAR-T targeting strategy based on metabolic glycoengineering (MGE) and bioorthogonal click chemistry. The core of this strategy is the bifunctional "molecular bridge" DBCO-CD19, which successfully transforms the complex targeting challenges posed by solid tumors into a clinically mature CD19 recognition paradigm. This strategy utilizes Ac4GalNAz for metabolic engineering, enabling tumor cells to exhibit a high density of artificial receptors with azide groups (-N3) on their surface. Subsequently, the DBCO moiety in DBCO-CD19 specifically covalently binds to these receptors via a copper-free click chemistry reaction. This "chemical anchoring" mode allows DBCO-CD19 to be broadly anchored to various tumor cell membranes, completely eliminating dependence on specific proteins on the natural tumor surface.

[0080] DBCO-CD19 was used to establish a highly efficient immune link between the CD19 antigen introduced on the surface of tumor cells and CD19 CAR-T cells, thereby mediating the specific recognition and killing of target cells (N3-K562 and N3-MDA-MB-231) by CAR-T cells. Furthermore, this strategy not only significantly enhanced the deep infiltration of CAR-T cells into 3D tumor spheroids but also mediated significant tumor regression in a zebrafish xenograft model. These experimental data confirm that regardless of the original surface markers of tumor cells, DBCO-CD19 can construct biologically functional CD19 antigens on cancer cell lines through a highly efficient click chemical reaction with -N3. These findings establish the reliability of DBCO-CD19 combined with metabolic sugar engineering as a broad-spectrum and highly versatile tumor marker method.

[0081] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A dual-functional universal targeted bridging molecule, DBCO-CD19, characterized in that: The bridging molecule was obtained by chemically modifying the tumor antigen CD19 protein with a dibenzocyclooctylene group.

2. The method for preparing the bridging molecule DBCO-CD19 according to claim 1, characterized in that: After dissolving CD19 protein, it is mixed with DBCO-PEG4-NHS at a molar ratio of 1:(30~50) and reacted at room temperature in the dark for 1~3 hours. After the reaction is completed, ultrafiltration is performed to remove unreacted DBCO-PEG4-NHS, thereby obtaining the bridging molecule DBCO-CD19.

3. The application of the bridging molecule DBCO-CD19 as described in claim 1 in enhancing the ability of CD19 CAR-T cells to target and eliminate tumor cells.

4. The use of the bridging molecule DBCO-CD19 according to claim 1 in the preparation of products that enhance the anti-tumor effect of CD19 CAR-T cells, characterized in that: The bridging molecule DBCO-CD19 enhances the anti-tumor effect of CD19 CAR-T cells by converting a highly heterogeneous tumor antigen profile into a single classical CD19 target, thereby inducing a strong immune recognition and killing between CD19 CAR-T cells and different malignant tumor cells.

5. The use of the bridging molecule DBCO-CD19 according to claim 1 in the preparation of a broad-spectrum antitumor product that is independent of natural target antigens.

6. A universal CAR-T cell anti-tumor composition or kit, characterized in that: It includes the bridging molecule DBCO-CD19 as described in claim 1.

7. The antitumor composition or kit according to claim 6, characterized in that: It also contains metabolic markers such as the carbohydrate precursor Ac4GalNAz and CD19 CAR-T cells.

8. The antitumor composition or kit according to claim 6 or 7, characterized in that: The concentration of the bridging molecule DBCO-CD19 is 1~50 μg / mL.

9. A method for universal CAR-T cell targeting and recognition of tumor cells for non-disease treatment and diagnosis purposes, characterized in that: The bridging molecule DBCO-CD19 described in claim 1 is anchored on the surface of metabolically labeled tumor cells expressing azide groups via a copper-free click reaction; immune cells achieve deep infiltration of solid tumors or 3D tumor sphere models and specific clearance of target tumor cells by binding to the CD19 antigen of the bridging molecule.

10. The method according to claim 9, characterized in that: The pairing groups for the copper-free click reaction are N3 and DBCO; the classical tumor antigen of the bridging molecule is CD19 protein; and the immune cells are CAR-T cells, CAR-NK cells, or CAR-M cells.

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