Graphene quantum dot and nano-antibody coupled antibody drug conjugate, and preparation method and application thereof

Abstract

The application discloses an antibody drug conjugate coupled by graphene quantum dots and nanobodies as well as a preparation method and application thereof. Through a precise covalent coupling strategy, the graphene quantum dots are coupled with anti-CEACAM6 nanobodies to obtain a functional synergistic targeted antibody drug conjugate for resisting tumors. The antibody drug conjugate has good hydrophilicity and good dispersibility in an aqueous solution, and is a strict single-atom-layer graphene quantum dot. After the antibody drug conjugate is co-incubated with cells, the cells are irradiated by a light source with a specific wavelength to make the GQD-NAB generate a large amount of active oxygen in the cells, significantly increase the toxicity to tumor cells, effectively realize the targeting of CEACAM6 positive tumors, and have more significant and safe anti-tumor effects. The antibody drug conjugate can be applied to the preparation of a photodynamic anti-tumor drug, and has important application prospects in actual clinical treatment of cancers.

Description

Technical Field

[0001] This invention relates to compounds conjugated with nanomaterials and nanoantibodies, particularly to compounds conjugated with graphene quantum dots and nanoantibodies, their preparation methods, and their application in the preparation of antitumor drugs or formulations, belonging to the field of antibody-drug conjugates and their applications. Background Technology

[0002] Currently, cancer treatment still faces three major clinical bottlenecks: First, traditional chemotherapy drugs, lacking specific recognition capabilities, exhibit indiscriminate cytotoxicity, often causing irreversible damage to normal tissues. Second, some nanomedicines still in the research and development stage suffer from significant batch-to-batch variations due to structural heterogeneity, severely limiting their clinical translation and application. Even targeted small molecule drugs or monoclonal antibodies are prone to drug resistance due to gene mutations or phenotypic changes in tumor cells, with resistance rates as high as 60%–80% in patients with advanced cancer. Third, treatment and monitoring are disconnected, making it impossible to track the dynamic effects of drugs on the tumor site in real time, resulting in treatment adjustments lagging behind tumor progression. These shortcomings collectively constitute a vicious cycle of "significant toxicity – limited efficacy – lagging monitoring," becoming the primary obstacle to the development of precision cancer treatment. Therefore, developing novel nanoplatforms that combine precise targeting, controlled release, real-time monitoring, and synergistic therapy has become an important direction for breaking through the current bottlenecks in cancer treatment.

[0003] The emergence of single-molecule graphene quantum dots (GQDs) provides a key breakthrough in overcoming the core defects of traditional nanomaterials. Compared with traditional nanomaterials, single-molecule GQDs have a well-defined chemical structure and a definite molecular weight, thereby eliminating batch-to-batch variability caused by structural heterogeneity at the source and ensuring the stability of drug efficacy and controllable toxicity. Simultaneously, their size is much smaller than traditional nanoparticles (typically less than 10 nm), enabling them to effectively penetrate the dense stromal barrier of tumors, achieving uniform drug distribution deep within the tumor parenchyma, and are easily cleared by the kidneys, reducing the risk of systemic accumulation. Furthermore, single-molecule GQDs exhibit high reactive oxygen species (ROS) yields, which can efficiently induce tumor cell apoptosis in photodynamic therapy (PDT); their stable fluorescence emission properties can also be used for real-time imaging of tumor sites, tracking drug distribution and treatment response, truly achieving "therapeutic integration." These advantages make single-molecule GQDs an ideal carrier for constructing modular, therapeutic nanosystems.

[0004] Antibody-drug conjugates (ADCs) represent a promising avenue for cancer therapy, enabling the selective delivery of highly cytotoxic payloads to tumor sites. However, fully realizing the potential of this platform requires addressing several clinical challenges through innovative molecular design, such as drug resistance, tumor heterogeneity, and treatment-related adverse reactions. The efficacy of targeted delivery systems is highly dependent on the targeting ability of the ligand. While traditional monoclonal antibodies (mAbs) possess high specificity, their large molecular weight (approximately 150 kDa) and complex structure (containing heavy and light chains) limit their tumor penetration, complicate manufacturing processes, increase costs, and may trigger immune-related adverse reactions. These factors restrict their application in deep treatment of solid tumors. In contrast, nanobodies, or single-domain antibodies (sdAbs), derived from the variable region (VHH) of alpaca heavy chain antibodies, exhibit several significant advantages. First, their simple structure and lack of light chains, with a molecular volume only about 1 / 10 that of traditional antibodies, not only make them easier to penetrate tumor tissues but also significantly reduce the difficulty and cost of expression and purification. Second, nanobodies possess higher affinity and thermal stability, enabling them to bind to target antigens efficiently and specifically, reducing the off-target risk caused by non-specific binding. Third, they exhibit extremely low immunogenicity and minimal toxicity, effectively avoiding the immunotoxicity commonly found in traditional antibodies, resulting in higher clinical safety. Furthermore, the N-terminus and C-terminus of nanobodies are easily modified, facilitating covalent conjugation with various carriers. These characteristics make nanobodies an ideal targeting tool for precision oncology. Summary of the Invention

[0005] One objective of this invention is to provide antibody-drug conjugates that combine graphene quantum dots with nanobodies; A second objective of this invention is to improve a method for preparing antibody-drug conjugates composed of graphene quantum dots and nanoantibodies; A third objective of this invention is to use the antibody-drug conjugate described above in the preparation of antitumor drugs.

[0006] The above-mentioned objectives of the present invention are mainly achieved through the following technical solutions: One aspect of the present invention provides an antibody-drug conjugate composed of graphene quantum dots and nanobodies, the structural formula of which is shown below:

[0007] Where R1 is ; The term refers to an anti-tumor nanobody; preferably, the anti-tumor nanobody is an anti-CEACAM6 single-domain antibody.

[0008] Another aspect of the present invention provides a method for preparing antibody-drug conjugates of graphene quantum dots and nanobodies, comprising: (1) Graphene quantum dots with edge-connected linkers were obtained by modifying graphene quantum dots (GQD-NH2) with linkers; (2) Graphene quantum dots with edge-connected linkers are covalently coupled with anti-tumor nanoantibodies to obtain antibody-drug conjugates of graphene quantum dots and nanoantibodies.

[0009] In a preferred embodiment of the present invention, the linker is preferably a straight-chain alkane NH2-(CH2)8-NH2; In a preferred embodiment of the present invention, the method for obtaining graphene quantum dots with edge-linked linkers by modifying graphene quantum dots (GQD-NH2) with linkers in step (1) includes: mixing PGQD and sodium methoxide in toluene to obtain a mixed solution; adding a toluene solution of 1,8-diaminooctane dropwise to the mixed solution under nitrogen protection; carrying out the reaction at a reaction temperature of 100°C; after the reaction is completed, removing the toluene solvent from the reaction system, dispersing the remaining residue in deionized water, dialyzing it using a dialysis bag with a molecular weight cutoff of 500 Da, and then freeze-drying it.

[0010] In a preferred embodiment of the present invention, the covalent coupling reaction in step (2) can be an amidation reaction or a click chemistry reaction; more preferably, the covalent coupling reaction includes: adding GQD-NH2 to the antitumor nanobody solution and reacting at room temperature; the room temperature can be 15-30℃, preferably 25℃. In a preferred embodiment of the present invention, the antitumor nanobody is preferably an anti-CEACAM6 single-domain antibody, and the anti-CEACAM6 single-domain antibody is preferably the single-domain antibody NBC10 or single-domain antibody NBC25 disclosed in the invention entitled "Anti-CEACAM6 Single-Domain Antibody, Humanized Single-Domain Antibody and its Fc Fusion Protein and Application" with publication number CN 112409485 A; wherein the amino acid sequences of the three complementarity-determining regions of the single-domain antibody NBC10 are shown in SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No. 3 respectively; the amino acid sequence of the single-domain antibody NBC10 is shown in SEQ ID No. 7; the amino acid sequences of the three complementarity-determining regions of the single-domain antibody NBC25 are shown in SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 6 respectively; preferably, the amino acid sequence of the single-domain antibody NBC25 is shown in SEQ ID No. 8.

[0011] The graphene quantum dots (GQDs) described in this invention are nanomaterials known to those skilled in the art. They are zero-dimensional carbon nanomaterials composed of graphene sheets with a lateral dimension of less than 100 nanometers, exhibiting unique quantum confinement effects, boundary effects, and excellent fluorescence properties. GQDs are graphene sheets with a lateral dimension of less than 100 nanometers, typically containing 1 to 10 graphene layers, with some ultra-small sizes less than 5 nanometers. The graphene quantum dots described in this invention can be purchased through various commercial channels or prepared by referring to the methods disclosed in relevant literature (such as CN 118359500A). Furthermore, graphene quantum dots obtained through various methods are applicable to this invention.

[0012] Another aspect of the present invention involves applying the antibody-drug conjugate to the preparation of photodynamic drugs or formulations for treating tumors.

[0013] This invention utilizes a precise covalent conjugation strategy (such as amide bonds or click chemistry) to combine single-molecule GQD with an anti-CEACAM6 (carcinoembryonic antigen-associated cell adhesion molecule 6) nanobody to construct a synergistic targeted therapy system, achieving a deep fusion of the advantages of both: On the one hand, the nanobody provides excellent active targeting capability, guiding GQD to specifically accumulate in CEACAM6-positive tumor regions, thereby overcoming the off-target toxicity of traditional therapies and the insufficient targeting efficiency of conventional nanomaterials; on the other hand, the nanobody's superior tumor penetration capability assists GQD in overcoming the dense stroma barrier, improving the limitations of traditional nanomedicines in deep tumor penetration. Furthermore, the well-defined structure of single-molecule GQD ensures the stability of the conjugation system, and its high ROS yield can significantly enhance the efficiency of PDT in the tumor local area, exhibiting an anti-tumor effect superior to traditional strategies. Meanwhile, the targeted inhibition of CEACAM6 by nanobodies can achieve a dual synergy of "killing tumors" and "inhibiting drug resistance", effectively reducing the risk of drug resistance caused by single therapy; and the inherent fluorescence emission characteristics of single-molecule GQD enable real-time monitoring of the treatment process, solving the problem of the disconnect between diagnosis and treatment.

[0014] The antibody-drug conjugate (GQD-NAb) provided by this invention has good hydrophilicity and dispersibility in aqueous solution, and is a strictly monolayer graphene quantum dot. After co-incubation with cells, it is irradiated with a specific wavelength of light to generate a large amount of ROS (reactive oxygen species) in the cells, which increases the toxicity to tumor cells, achieves CEACAM6-positive tumor targeting, and has a more significant and safer anti-tumor effect. It can be used to prepare photodynamic anti-tumor drugs and has great application prospects in actual clinical cancer treatment. Attached Figure Description

[0015] Figure 1 This is a flowchart of the synthesis process of GQD-NAb.

[0016] Figure 2 This is the mass spectrum of GQD-NAb.

[0017] Figure 3 This is a TEM image of GQD-NH2.

[0018] Figure 4 The in vitro PDT effect of GQD-NAb was measured using white light, with illumination times of 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 seconds.

[0019] Figure 5 The UV-vis spectrum and PL fluorescence spectrum of GQD-NAb are shown (left: UV-vis; right: PL).

[0020] Figure 6 The results of the hTERT-HPNE toxicity test of GQD-NAb on normal cells.

[0021] Figure 7 The image shows intracellular fluorescence of PANC-1 cells co-cultured with GQD-NAb under a laser confocal microscope.

[0022] Figure 8 The results show the inhibitory effect of GQD-NAb light on PANC-1 tumor cells in the group and the control group.

[0023] Figure 9 This image shows the therapeutic effect of GQD-NAb on subcutaneous xenografts of PANC-1 cells in mice. Detailed Implementation

[0024] The present invention will be further described below with reference to specific experimental examples, and the advantages and features of the present invention will become clearer with the description. However, these experimental examples are merely exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but all such modifications and substitutions fall within the protection scope of the present invention.

[0025] Table 1. Main chemical reagents used in the following examples or test cases.

[0026] Note: No further treatment is required before use. The resistivity of the experimental water is 18.25 MΩ cm. -1 .

[0027] Example 1: Preparation and characterization of antibody-drug conjugates (GQD-NAb) conjugated with graphene quantum dots and nanobodies. 1. Preparation of linker-modified GQD-NH2 Linker-modified graphene quantum dots were obtained through precise covalent coupling. The specific preparation method included: mixing PGQD (Pure Grahene Quantum Dots; graphene quantum dots prepared according to the method disclosed in CN 118359500A, entitled "A Single-Layer Water-Soluble Graphene Quantum Dot and its Preparation Method and Use") (0.0154 mmol) with sodium methoxide (0.0154 mmol) in toluene (5 mL) to obtain a mixed solution; subsequently, under nitrogen protection, a toluene solution of 1,8-diaminooctane (0.24 mmol) (5 mL) was slowly added dropwise to the above mixed solution. After the addition was complete, the reaction system was heated to 100 °C and maintained at this temperature for 48 h. After the reaction was completed, toluene solvent in the system was removed by vacuum distillation; the remaining residue was dispersed in deionized water (10 mL) and dialyzed for 24 h using a dialysis bag with a molecular weight cutoff of 500 Da to obtain a yellow-brown solution. The solution was then freeze-dried to finally obtain a brown solid product—aminofunctionalized graphene quantum dots (GQD-NH2).

[0028] Linker-modified graphene quantum dots with the structure shown below were prepared:

[0029] 2. Linker-modified graphene quantum dots covalently coupled with nanobodies Take a concentration of 1 mg / mL -1 A 5 mL nanobody (the nanobody being the single-domain antibody NBC10 disclosed in the invention titled "Anti-CEACAM6 Single-Domain Antibody, Humanized Single-Domain Antibody and its Fc Fusion Protein and Application", publication number CN 112409485 A, with its amino acid sequence shown in SEQ ID No. 7) was taken out of a -80 ℃ freezer and thawed and reconstituted according to the conventional low-temperature thawing procedure; 5 mg of GQD-NH2 was added to the reconstituted antibody solution, and the reaction was carried out at room temperature; the reaction was stirred for 2 h, and the GQD-NAb antibody-drug conjugate (GQD-NAb) was obtained after the reaction was completed. Figure 1 This is a flowchart of the synthesis process of GQD-NAb.

[0030] 3. Characterization of antibody-drug conjugate (GQD-NAb) Testing revealed that the prepared antibody-drug conjugate (GQD-NAb) product had a uniform particle size distribution and good optical properties.

[0031] Figure 2 The mass spectrum of the synthesized GQD-NAb is shown. Figure 3 The image shows a TEM image of the synthesized GQD-NH2.

[0032] Experimental Example 1: In vitro PDT efficacy assay of antibody-drug conjugate (GQD-NAb) 1. Experimental Methods 2',7'-dichlorofluorescein diacetate (DCFH-DA) was dissolved in a dimethyl sulfoxide (DMSO) solvent system to obtain a mixture. A 1 mol / L solution was then added to the resulting mixture. -1 An aqueous solution of sodium hydroxide was stirred continuously in a light-protected environment for 2 hours; after the reaction was complete, a 1 mol / L solution was added dropwise. -1 The pH of the system was adjusted by adding hydrochloric acid aqueous solution until the pH of the solution stabilized at 7.4, thus obtaining 2',7'-dichlorofluorescein (DCFH) stock solution.

[0033] The prepared DCFH stock solution was sealed and stored in a light-protected, frozen environment for subsequent experiments. 250 μL of the DCFH stock solution was mixed with 2.75 mL of GQD-Nab (prepared in Example 1) solution. The prepared mixed samples were then placed in different wavelengths and at an optical power density of 20 mW / cm². -2 The system is irradiated under a laser light source, and the qualitative and quantitative analysis of reactive oxygen species generated within the system is performed using fluorescence spectroscopy detection equipment.

[0034] 2. Experimental Results Figure 4 To demonstrate the PDT effect of GQD-NAb in vitro, white light was used as the light source, and the illumination time was 0, 10, 20, 30, 40, 50, 60, 70, 80, and 90 seconds. The in vitro ROS release results of the GQD-NAb aqueous solution showed that the ROS content released from the GQD-NAb aqueous solution gradually decreased with increasing illumination time. Compared with no illumination, the ROS content was highest at 70 seconds of illumination.

[0035] Experimental Example 2: UV-vis and PL fluorescence spectroscopy determination of antibody-drug conjugate (GQD-NAb) 1. Experimental Methods GQD-NAb (prepared in Example 1) was prepared to a concentration of 0.1 μg / mL. -1The concentration of GQD-NAb was determined, and the sample was placed in a quartz double-pass cuvette. The absorption between 200 nm and 600 nm was measured using a UV-3600 (Shimadzu, Japan) UV-Vis spectrometer, and absorption curves were plotted. Separately, a GQD-NAb solution of the same concentration was prepared, placed in a quartz four-pass cuvette, and its polarity was measured using an RF-6000 (Shimadzu, Japan) fluorescence emission spectrometer. Emission spectra at excitation wavelengths from 450 nm to 550 nm were obtained, and PL fluorescence spectra were plotted.

[0036] 2. Experimental Results Figure 5 The UV-vis spectrum and PL fluorescence spectrum of GQD-NAb are shown (left: UV-vis; right: PL).

[0037] Experimental Example 3: Antibody-Drug Conjugate (GQD-NAb) for Photodynamic Therapy (PDT) Antitumor Assay The antibody-drug conjugate GQD-NAb (prepared in Example 1) was co-incubated with cells to induce the cells to generate ROS under light, thereby increasing the toxicity to tumor cells and achieving photodynamic (PDT) anti-tumor activity.

[0038] I. Verification methods for the cytotoxicity of GQD-NAb Step 1: The GQD-NAb prepared in Example 1 was prepared in DMEM medium to concentrations of 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, 0.19, and 0.1 µg / mL. -1 The solution.

[0039] The second step is to seed hTERT-HPNE cells (human normal pancreatic duct epithelial cells) into 96-well cell culture plates at a density of 4000-5000 cells per well. After culturing at 37°C, 5% CO2, and saturated humidity for 24 h to allow them to adhere, the culture medium is removed.

[0040] The third step involves adding the prepared GQD-NAb solutions of different concentrations into 96-well plates and co-culturing them with the cells at 37°C for 24 h. The blank control group consists of 100 µL of serum-free culture medium.

[0041] Step 4: After cultivation, add 5 mg mL to each well. -1 Add 20 µL of MTT and continue incubation for 4 hours. Remove the supernatant and add 150 µL of DMSO to each well. Mix well and measure the absorbance at 490 nm using a microplate reader. Perform 6 parallel measurements for each experiment.

[0042] The test results are Figure 6As shown, the MTT measurement results indicate that under light-free conditions, the concentration of GQD-NAb at 25 µg / mL... -1 It is non-toxic.

[0043] II. Cellular uptake and ROS detection assay of GQD-NAb Step 1: [The text appears to be incomplete and contains 1×10...] 5 One mL of DMEM medium per cell was seeded into a 35 mm glass bottom confocal culture dish (MatTek). After culturing under standard conditions (37℃, 5% CO2) for 24 h, the medium was removed.

[0044] Step 2: Add 1 mL of solution containing 10 µg mL -1 Fresh GQD-NAb culture medium was co-cultured at 37°C for 4 hours. Cells were then carefully washed three times with PBS to remove any GQD-NAb that had not been taken up by the cells. The blank control group consisted of 1 mL of serum-free culture medium.

[0045] Step 3: Add 1 mL of 10 µM DCFH solution (prepared in Example 1), incubate in an incubator for 15 min, irradiate with a 420 / 470 nm light source for 1 min, incubate again in an incubator for 15 min, and then carefully wash the cells three times with PBS to remove excess DCFH dye.

[0046] Step 4: Use 1 mL of 10 µg mL of [unspecified solution] to [unspecified solution]. -1 Cell nuclei were stained with Hoechst 33258, then incubated in an incubator for 5 min. Cells were then carefully washed three times with PBS to remove excess Hoechst dye.

[0047] Fifth, finally, a confocal laser scanning microscope (Nikon Ni-EC2+, Japan) was used to observe the uptake of nanoparticles by cells and the release of intracellular ROS.

[0048] Figure 7 The images show intracellular fluorescence in PANC-1 cells co-cultured with GQD-NAb under laser confocal microscopy. Based on the laser confocal images of GQD-NAb uptake and intracellular ROS release, it is evident that GQD-NAb accumulates in the nuclei of PANC-1 cells; and the ROS generated by GQD-NAb after illumination is distributed intracellularly.

[0049] III. Intracellular photodynamic effect assay of GQD-NAb Step 1: PBS, GQD-NH2, Ab (a single nanobody with the amino acid sequence shown in SEQ ID No. 7), VP6 (a fusion protein of the single nanobody shown in SEQ ID No. 7 and human IgG1-Fc), and GQD-NAb were prepared in DMEM medium to concentrations of 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, and 0.19 mol / mL. -1 The solution.

[0050] The second step is to seed PANC-1 cells into 96-well cell culture plates at a density of 4000-5000 cells per well. After incubation at 37°C, 5% CO2, and saturated humidity for 24 hours to allow them to adhere, the culture medium is then removed.

[0051] The third step involves adding the prepared GQD-NAb solutions of different concentrations into 96-well plates and incubating them with cells at 37°C for 4 hours. After irradiating with white light for 1 minute, the plates are then placed in an incubator for 20 hours. The blank control group consists of 100 µL of serum-free culture medium.

[0052] Step 4: After incubation, add 5 mg mL to each well. -1 Add 20 µL of MTT and continue incubation for 4 h. Remove the supernatant and add 150 µL of DMSO to each well. Mix well and measure the absorbance at 490 nm using a microplate reader. Perform 6 parallel measurements for each experiment.

[0053] The test results are Figure 8 As shown in the experimental results, compared with the control groups (PBS, GQD-NH2, Ab, VP6, GQD-NAb unilluminated group), the cell viability in the GQD-NAb-L group after illumination significantly decreased with increasing GQD-NAb concentration. Under white light illumination, cell viability could be increased at 0.78 µL / mL. -1 At the drug concentration, a cell activity inhibition rate of 80% was achieved, indicating that GQD-NAb has high PDT efficiency.

[0054] IV. Photodynamic Effect of GQD-NAB on Pancreatic Cancer in Mice The first step involves mixing PBS, GQD-NH2, Ab (a single nanobody with the amino acid sequence shown in SEQ ID No. 7), VP6 (a fusion protein of a single nanobody with the amino acid sequence shown in SEQ ID No. 7 and human IgG1-Fc), and GQD-NAb (100 μL, 150 μmol / L). -1 ) was injected into female C-NKG mice via tail vein (5 mg kg) -1(n=5), the dosing frequency is once every 3 days, for a total of 5 times.

[0055] The second step involved the light group receiving white light exposure (0.5 W cm⁻¹) every four days. -2 (3 min).

[0056] The third step involved recording the body weight and tumor growth curve of mice in each group every two days for 14 days.

[0057] The fourth step involves measuring the two-dimensional tumor volume using vernier calipers and then applying the formula... Perform the calculation.

[0058] The test results are as follows Figure 9 As shown, the analysis of ex vivo tumor weight revealed that the GQD-NAb+L treatment group (Note: L is an abbreviation for "Light," indicating that this experimental group was treated with light) exhibited the strongest tumor-suppressing effect, with a tumor weight inhibition rate of 80.78%, significantly higher than the VP6 group (30.73%), GQD-NAb (48.27%), and GQD-NH2+L group (Note: L is an abbreviation for "Light," indicating that this experimental group was treated with light) (46.18%). Post-treatment anatomical tumor photographs showed that the GQD-NAb+L group had the smallest tumor volume. During treatment, dynamic monitoring of tumor volume showed that the tumors in the PBS and GQD-NH2 groups continued to grow, while the tumor volume in the GQD-NAb+L treatment group began to shrink significantly from day 8. The ex vivo tumor volume measurement results further confirmed that this group had the most significant tumor-suppressing effect.

Claims

1. An antibody-drug conjugate composed of graphene quantum dots and nanobodies, characterized in that, The structural formula of the antibody-drug conjugate is shown below: Where R1 is ; This represents anti-tumor nanobodies.

2. The antibody-drug conjugate according to claim 1, characterized in that, The anti-tumor nanobody is an anti-CEACAM6 single-domain antibody, and its amino acid sequence is shown in SEQ ID No. 7 or SEQ ID No.

8.

3. A method for preparing the antibody-drug conjugate according to claim 1 or 2, characterized in that, include: (1) Graphene quantum dots with edge-connected linkers were obtained by modifying graphene quantum dots with linkers; (2) Graphene quantum dots with edge-connected linkers are covalently coupled with anti-tumor nanoantibodies to obtain antibody-drug conjugates of graphene quantum dots and nanoantibodies.

4. The method according to claim 3, characterized in that, The linker is a straight-chain alkane NH2-(CH2)8-NH2.

5. The method according to claim 3, characterized in that, The method for obtaining graphene quantum dots with edge-connected linkers by modifying graphene quantum dots with linkers in step (1) includes: mixing PGQD and sodium methoxide in toluene to obtain a mixed solution; adding a toluene solution of 1,8-diaminooctane dropwise to the mixed solution under nitrogen protection; carrying out the reaction at a reaction temperature of 100°C; after the reaction is completed, removing the toluene solvent from the reaction system, dispersing the remaining residue in deionized water, dialyzing it using a dialysis bag with a molecular weight cutoff of 500 Da, and then freeze-drying it.

6. The method according to claim 3, characterized in that, The covalent coupling reaction is an amidation reaction or a click chemistry reaction; preferably, the covalent coupling reaction includes: adding GQD-NH2 to the antitumor nanobody antibody solution and reacting at room temperature; the room temperature is preferably 15-30°C, more preferably 25°C.

7. The method according to claim 3, characterized in that, The anti-tumor nanobody is an anti-CEACAM6 single-domain antibody, and its amino acid sequence is shown in SEQ ID No. 7 or SEQ ID No.

8.

8. Use of the antibody-drug conjugate of claim 1 or 2 in the preparation of a medicament or formulation for treating tumors.

9. The use according to claim 8, characterized in that, The drug or preparation mentioned is a photodynamic drug or preparation.

10. A pharmaceutical composition for treating tumors, characterized in that, It consists of a therapeutically effective amount of the antibody-drug conjugate of claim 1 or 2 and an excipient or carrier for pharmaceutical formulation.

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