PD-L1 / TGF-beta / STING triple targeting antibody coupling drug as well as preparation method and application thereof

Abstract

The invention discloses a PD-L1 / TGF-beta / STING triple targeting antibody coupling drug as well as a preparation method and application thereof, and belongs to the field of tumor immunotherapy. The ADC structure is Ab-[L-D] n, Ab is a bispecific antibody YM101 targeting PD-L1 and TGF-beta, L is a cleavable connexon, and D is an STING agonist. The preparation method comprises the following steps: carrier molecule deprotection, linker functionalization, linker-load coupling, antibody sulfhydrylation modification, coupling reaction and affinity chromatography purification. The ADC can increase the number of CXCL16 < + > macrophages, dendritic cells and CXCR6 < + > T cells infiltrated in tumors, effectively reverses the drug resistance of a TGF-beta * PD-L1 bispecific antibody, has a strong anti-tumor effect and no obvious toxic or side effect, and has a good application prospect in preparation of anti-tumor drugs.

Description

Technical Field

[0001] The present invention belongs to the field of tumor immunotherapy, and specifically relates to a PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate, and a preparation method and application thereof. Background Art

[0002] The advent of immune checkpoint inhibitors (ICIs), particularly those targeting the PD-1 / PD-L1 axis, has revolutionized cancer treatment. Despite this success, many patients fail to respond to ICI therapy due to resistance. In these cases, PD-1 / PD-L1 signaling may not be the primary mechanism driving tumor immune evasion, suggesting that other immune pathways are also affected. Addressing these imbalances within the tumor microenvironment (TME) is crucial for restoring effective anti-tumor immunity. Combining ICIs with other therapeutic strategies has emerged as a promising approach to enhance their efficacy and overcome resistance. Numerous studies have identified drugs that exhibit synergistic effects when combined with PD-1 / PD-L1 inhibitors. However, these combination strategies have met with limited success. For example, the combination of the STING agonist ADU-S100 with PD-1 blockade resulted in only a 10% overall response rate in patients with advanced tumors, highlighting the need for more effective approaches. Understanding the molecular mechanisms underlying ICI combination therapy and the interplay of signaling pathways within the TME is crucial for advancing immunotherapy.

[0003] Transforming growth factor β (TGF-β) plays a dual role in cancer. During early tumor development, it inhibits tumor growth by regulating cell proliferation, apoptosis, and differentiation. However, in advanced tumors, mutations in TGF-β signaling often negate its tumor suppressor effects. Instead, TGF-β promotes tumor progression by inducing epithelial-mesenchymal transition (EMT), enhancing angiogenesis, and activating cancer-associated fibroblasts. Elevated TGF-β levels in the TME also lead to immune escape by suppressing T cell and NK cell activity, impairing antigen presentation by dendritic cells (DCs), and promoting the differentiation of immunosuppressive cells. High levels of TGF-β expression are associated with poor prognosis and resistance to immunotherapy, making TGF-β inhibition a potential strategy to improve the efficacy of ICIs.

[0004] BsAbs (bispecific antibodies) are antibodies that can simultaneously bind to two different antigens or two different epitopes on the same antigen. This property gives BsAbs unique advantages in tumor immunotherapy, by redirecting effector cells (such as T cells, NK cells, macrophages, and monocytes) to tumor cells and enhancing their ability to kill tumor cells in a non-MHC-restricted manner. Furthermore, BsAbs not only provide an effective connection between therapeutics (e.g., immune effector cells, radionuclides) and targets (e.g., tumor cells), but also can simultaneously block two different cancer signaling pathways, such as EGFR×HER2 bispecific antibodies and EGFR×c-MET bispecific antibodies. As of March 2025, 13 BsAbs have been approved globally, including blinatumomab, candonilimab, emicizumab, and amivantamab. These bispecific antibodies have demonstrated clinical efficacy superior to single-target antibodies in the treatment of tumors, hemophilia A, diabetes, Alzheimer's disease, and ophthalmological diseases. Although a number of BsAbs have been approved for use, the vast majority are still in various stages of clinical development. Compared to the combination of two monofunctional antibodies, the primary advantage of BsAbs lies in their ability to simultaneously target two different antigens or two distinct epitopes on a single antigen. This significantly enhances specific binding to tumor cells and improves therapeutic efficacy. Furthermore, by improving therapeutic targeting, BsAbs also reduce the risk of side effects during treatment. These advantages indicate that BsAbs hold broad application prospects in future medical treatments.

[0005] To combat TGF-β-mediated immunosuppression and resistance to immune checkpoint blockade, the inventors previously developed YM101 (patent CN112851819B), a TGF-β×PD-L1 BsAb. Although YM101 has shown greater efficacy than PD-L1 inhibitors alone, some tumors remain unresponsive. This challenge is also common in second-generation PD-L1 antibodies such as M7824 and SHR-1701, highlighting the complexity of TGF-β signaling and the need to explore the mechanisms of treatment failure. Further studies in the present invention have shown that there is a strong synergistic effect between YM101 and STING agonists. In multiple preclinical tumor models, the combination with STING agonists significantly inhibited tumor growth and prolonged survival in mice treated with YM101. Logically, STING agonists initiate anti-tumor immune responses early in the cancer-immunity cycle, enhancing adaptive immunity. In addition, based on this discovery, the present invention developed the first TGF-β×PD-L1-STING antibody-drug conjugate (ADC), which combines TGF-β×PD-L1BsAb with a STING agonist connected by a cleavable linker. This new ADC targets all three pathways simultaneously, providing a strategic advantage over traditional multi-drug combinations and has the potential to improve treatment outcomes for cancer patients in the future. Summary of the Invention

[0006] In response to the deficiencies in the prior art, the present invention innovatively developed a PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate, its preparation method, and application. The present invention found that weakened STING signaling in tumors may be the main cause of TGF-β×PD-L1BsAb resistance, and that recruiting CXCR6+ T cells through STING agonists can reverse resistance. Based on this theory, the present invention developed the STING / TGF-β / PD-L1 antibody-drug conjugate (ADC) Y101S, which targets tumor tissues, and verified its anti-tumor effect in multiple mouse tumor-bearing models.

[0007] To achieve the above object, the present invention is implemented through the following technical solutions:

[0008] In a first aspect, the present invention provides a PD-L1 / TGF-β / STING triple-targeting antibody-drug conjugate, the structure of which is shown in formula (I):

[0009] Ab-[LD] n (I)

[0010] Wherein, Ab is the bispecific antibody YM101 that binds to PD-L1 and TGF-β; L is a cleavable linker; D is a STING agonist; n is the number of Ds connected to Ab via the cleavable linker L, and n is a positive integer; "-" is a chemical bond.

[0011] As a further optimized solution of the present invention, the STING agonist includes MSA-2.

[0012] In a second aspect, the present invention provides a method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate, comprising the following steps:

[0013] (1) Deprotecting the carrier molecule containing the cleavable linker unit to obtain intermediate I;

[0014] (2) coupling the intermediate I with a bifunctional linker containing a maleimide group in the presence of an organic base to obtain the intermediate II;

[0015] (3) condensing the STING agonist with intermediate II in the presence of a condensing agent and a catalyst to form a cleavable linker-drug complex;

[0016] (4) sulfhydryl modification of the anti-PD-L1 and TGF-β bispecific antibody YM101;

[0017] (5) performing a coupling reaction between the cleavable linker-drug complex obtained in step (3) and the thiol-ylated antibody obtained in step (4);

[0018] (6) The target ADC product was obtained by affinity chromatography purification.

[0019] As a further optimization scheme of the present invention, in step (1), the carrier molecule includes Fmoc-Val-Cit-PAB, and the deprotection reaction uses a mixed system of a piperidine alkaline reagent and a polar / aprotic solvent to remove the Fmoc protecting group.

[0020] As a further optimized solution of the present invention, in step (2), the bifunctional linker includes NHS-PEG4-Mal, the organic base includes DIPEA, and the molar ratio of the intermediate I to the bifunctional linker is (0.9-1):1.

[0021] As a further optimized solution of the present invention, in step (3), the molar ratio of the STING agonist to the intermediate II is (0.9-1):1, the condensing agent includes EDC.HCl, and the catalyst includes DMAP.

[0022] As a further optimized solution of the present invention, in step (4), the thiol-forming reagent includes Traut's reagent.

[0023] As a further optimization scheme of the present invention, in step (5), the molar ratio of the cleavable linker-drug complex to the thiol-modified antibody is 4:1

[0024] In a third aspect, the present invention provides a use of the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate in the preparation of anti-tumor drugs.

[0025] As a further optimized solution of the present invention, the drug is used to increase the number of CXCL16+ macrophages, dendritic cells and CXCR6+ T cells infiltrating the tumor.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) The ADC of the present invention has a strong anti-tumor effect, effectively reverses the drug resistance of TGF-β×PD-L1BsAb, and has no obvious toxic side effects. In experiments on multiple tumor-bearing mouse models (such as EMT-6, 4T1 and CT26 tumor-bearing mouse models), compared with the use of YM101 alone, the ADC drug Y101S has better efficacy, and no treatment resistance has occurred. It significantly inhibits tumor growth and prolongs the survival of mice. It has good in vivo safety and has no obvious damage to the liver function, kidney function, and heart function of mice. After multiple administrations to EMT-6 tumor-bearing mice, blood biochemical index detection showed that there was no significant change in the relevant organ function indicators, reducing the safety risks in clinical application.

[0028] (2) The ADC of the present invention can recruit CXCR6+ T cells and exert better anti-tumor activity. By promoting the increase in the number of CXCL16+ macrophages and dendritic cells in the tumor microenvironment, the recruitment of CXCR6+ T cells is enhanced. Because CXCR6+ T cells have strong tumor-killing activity, the body's anti-tumor immune response is enhanced, thereby enhancing the therapeutic effect of the drug.

[0029] (3) The ADC preparation method of the present invention is simple and easy to purify. During the synthesis process, the reaction conditions of each step are mild and the operation is conventional. For example, the deprotection reaction is carried out at room temperature using a piperidine / DMF mixture, and the subsequent coupling reaction is also carried out under common conditions such as room temperature or ice bath. The separation and purification of the product is achieved by standard experimental methods such as precipitation, washing, ultrafiltration centrifugation, desalting column and protein A column chromatography. These methods are easy to implement in both laboratory and industrial production, reducing production difficulty and cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1This is a graph showing the transcriptome sequencing results in Example 1 of the present invention. A represents the expression level of IFN-β (encoding gene Ifnb1), a core molecule of the STING pathway, in the drug-resistant group, and B represents the expression level of other STING pathway downstream cytokines in YM101-resistant tumors.

[0031] Figure 2 This figure shows the therapeutic effect of a STING agonist combined with YM101 in Example 2 of the present invention. A represents the change in tumor volume after inoculation with different drugs, B represents the change in the number of CXCL16+ macrophages and dendritic cells in the tumor microenvironment after treatment with a STING agonist combined with YM101, and C represents the change in the number of CXCR6+ T cells in the tumor microenvironment after treatment with a STING agonist combined with YM101.

[0032] Figure 3 This is a technical roadmap for preparing the PD-L1 / TGF-β / STING triple-targeted ADC in Example 3 of the present invention. A is a schematic diagram of linker-payload synthesis, and B is a schematic diagram of ADC synthesis.

[0033] Figure 4 This figure shows the anti-tumor effect of the ADC triple-targeted with PD-L1 / TGF-β / STING in EMT-6, 4T1, and CT26 tumor-bearing mouse models in Example 4 of the present invention. Figures A and B show the anti-tumor effect of different drugs inoculated in EMT-6 tumor-bearing mice, B show the anti-tumor effect of different drugs inoculated in 4T1 tumor-bearing mice, and C show the anti-tumor effect of different drugs inoculated in CT26 tumor-bearing mice.

[0034] Figure 5 This is a diagram of the ADC safety evaluation in Example 5 of the present invention.

[0035] Figure 6 This is a graph showing the therapeutic effect of ADC in Example 6 of the present invention, wherein: A represents the change in the number of CXCL16+ macrophages and dendritic cells infiltrating the tumor, and B represents the change in the number of CXCR6+ T cells infiltrating the tumor. DETAILED DESCRIPTION

[0036] Below with reference to embodiment, embodiment of the present invention is described in detail.It will be appreciated by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention.In the examples, if no specific techniques or conditions are indicated, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer of the reagents or instruments used is not indicated, it is a conventional product that can be purchased on the market.

[0037] Example 1: STING signaling attenuation causes TGF-β×PD-L1BsAb resistance

[0038] Female BALB / c mice aged 6-8 weeks were used to expand EMT-6 breast cancer cells and then inoculated with 5×10 4 One week after inoculation, when the tumor grew to about 100 mm 3 At 4 hr, mice were randomly divided into two groups and administered with equimolar amounts of hIgG (Isotype, Wuhan Youzhiyou Biotechnology) and YM101 (Y101-IGY, Wuhan Youzhiyou Biotechnology) intraperitoneally at doses of 6.6 mg / kg and 9 mg / kg, respectively. The doses were administered every other day for a total of 6 times. During this period, the tumor size was measured and calculated using the formula of length × width × width × 0.5. Finally, when the tumor volume exceeded 2500 mm 3 Mice were sacrificed at the end of the experiment. The average tumor volume of the hIgG group at the last measurement was used as the control. Individuals whose tumor volume decreased by less than 20% after YM101 treatment compared to the hIgG group were defined as drug-resistant, while those whose tumor volume decreased by more than 20% were defined as treatment-sensitive.

[0039] Fresh tumor tissue was taken out and washed in PBS. Then, the washed tumor tissue was placed in a sterile tube and quickly placed in a liquid nitrogen tank for rapid cooling. After grinding the tumor tissue with liquid nitrogen, the RNA in the tissue was extracted using the Trizol method. Subsequently, absolute quantitative transcriptome sequencing was completed using the services of Wuhan Kangce Technology Co., Ltd., and the reference genome was Mus_musculus.GRCm38. Finally, R software and edgeR package were used for differential gene analysis, and the screening criteria were set as genes with a differential expression fold greater than 2 and a P value less than 0.05. The results showed that molecules related to the STING pathway were significantly downregulated in the YM101-resistant group ( Figure 1 ), among which the core molecule of the STING pathway, IFN-β (encoding gene Ifnb1), was lost in the drug-resistant group ( Figure 1 A), other STING pathway downstream cytokines were also significantly decreased in YM101-resistant tumors ( Figure 1 B), the results suggest that weakened STING pathway activation may be an important factor in TGF-β×PD-L1BsAb resistance.

[0040] Example 2: Increasing CXCL16 expression through STING agonists can recruit CXCR6+ T cell infiltration and reverse TGF-β×PD-L1BsAb resistance

[0041] The present invention tested whether STING agonists can enhance the anti-tumor effect of YM101 and reverse its drug resistance in EMT-6 tumor-bearing mice. After BALB / c mice were loaded with EMT-6 tumors according to the above method, when the tumors grew to about 100 mm 3At the same time, the mice were randomly divided into 4 groups and treated with hIgG, YM101, MSA-2 (HY-136927, MCE), and YM101 combined with MSA-2. Among them, the doses of hIgG and YM101 were 6.6 mg / kg and 9 mg / kg, respectively, and MSA-2 was 50 mg / kg. hIgG and YM101 were injected intravenously as a single dose, and a total of 3 times were administered. MSA-2 was administered orally as a single dose, which was performed simultaneously with the first antibody injection. The tumor volume monitoring of mice was the same as above, and the results showed that the STING agonist significantly enhanced the therapeutic effect of YM101. The tumor volume of all mice in the MSA-2 combined with YM101 group showed a significant reduction (the volume reduction rate was greater than 20%), which effectively overcame the emergence of YM101 resistance ( Figure 2 A).

[0042] Subsequently, the present invention prepares single cell suspension in mouse tumor tissue and performs flow cytometric analysis. First, the mouse is killed to remove fresh tumor tissue, which is chopped after rinsing with PBS and incubated at 37 ° C for 1 hour with a digestive solution containing collagenase B (0.5 mg / ml, 11088807001, Roche) and hyaluronidase (0.5 mg / ml, abs47014926, Absin) to decompose the tissue. Single cell suspension is obtained by filtration through a cell strainer (352340, Corning), followed by red blood cell lysis solution (C3702, Biyuntian) treatment, and live cells are labeled with a dead dye (1: 200, 65-0865-18, Thermo). Then, the cells are subjected to Fc receptor blocking (1: 100, 14-0161-86, Thermo), flow antibody staining (specific surface staining antibody dosage and article number are shown in Table 1) and other steps. Finally, the treated cells were resuspended in PBS containing 1% FBS and analyzed using a flow cytometer (FACSCELESTA, BD). The results showed that the STING agonist combined with YM101 treatment effectively promoted the number of CXCL16+ macrophages and dendritic cells in the tumor microenvironment ( Figure 2 B), thereby enhancing the recruitment of CXCR6+ T cells ( Figure 2 C) Classical tumor immunology holds that CXCR6+ T cells are tumor-resident T cells with strong tumor-killing activity.

[0043] Table 1 Flow cytometry antibodies used in mouse tumor microenvironment detection

[0044] Antibody name Item No. factory Use concentration MouseCD11bFITCantibody 11-0112-82 Thermo 1:200 MouseCD11cAPCantibody 117310 BioLegend 1:100 MouseCD16 / 32 blocking antibody 14-0161-86 Thermo 1:100 MouseCD3BUV395antibody 563565 BD 1:100 MouseCD45-BV510antibody 563891 BD 1:100 MouseCD8BV421antibody 100753 BD 1:100 MouseCD86FITCantibody 11-0862-85 Thermo 1:200 MouseCXCL16PEantibody 566740 BD 1:100 MouseCXCR6APCantibody 151106 BioLegend 1:100 MouseF4 / 80BV421antibody 123131 BioLegend 1:100 Mouse MHC-IIBV786 antibody 417-5321-82 Thermo 1:100

[0045] Example 3: Preparation of ADC with triple targeting of PD-L1 / TGF-β / STING

[0046] like Figure 3 As shown in Figure 1, 200 mg of Fmoc-Val-Cit-PAB (Y-PE-4775, Shaanxi Xinyan Bomei) was dissolved in a mixture of piperidine (400 μl) and DMF (2 ml) (volume ratio 1:4) and stirred at room temperature for 5 hours. After the reaction was completed, the reaction solution was poured into cold ether (50 mL) to precipitate the product. The solid was collected by suction filtration and washed three times with cold ether (10 mL each). The product was vacuum dried to obtain intermediate 1 (Int1).

[0047] Subsequently, Int1 (70 mg, 0.184 mmol) was dissolved in DMF (1 mL) and stirred, and then NHS-PEG4-Mal (89.8 mg, 0.203 mmol) (Y-PE-2017, Shaanxi Xinyan Bomei) and DIPEA (64.3 μL, 0.369 mmol) (D125806, Sigma) were added to the reaction solution. After stirring at room temperature for overnight, the reaction solution was poured into cold ether (10 mL) to precipitate the product, and the solid was collected by suction filtration and washed three times with cold ether (5 mL each). The product was dried in vacuo to obtain intermediate product 2 (Int2).

[0048] MSA-2 (25 mg, 0.085 mmol) and EDC.HCl (24.4 mg, 0.127 mmol) were dissolved in DMF (1 mL) and stirred at 0°C in an ice bath. Int2 (67.35 mg, 0.093 mmol) and DMAP (20.75 mg, 0.170 mmol) were then added, and the reaction was stirred at room temperature overnight. The reaction solution was poured into water (10 mL) to precipitate the product. The solid was collected by suction filtration and washed three times with ethyl acetate (10 mL each) and twice with dichloromethane to obtain the linker-payload complex.

[0049] YM101 (20 mg) and Traut's reagent (20 eq) (26101, Thermofisher) were mixed in PBS buffer (pH = 7.4) to prepare a thiol-functionalized primary antibody. Unreacted excess reagents were then removed using an Amicon ultrafiltration centrifuge tube. The thiol-functionalized primary antibody and linker-payload (40 ug) were dissolved in a solution containing 10% DMSO and 10% PEG300 at a molar ratio of 1:4 and stirred at room temperature for 2 hours. After the reaction, the unbound excess linker-payload was removed by a desalting column equilibrated with PBS. The resulting antibody-drug conjugate (ADC) was then loaded onto a 1 mL protein A column (Cytiva) pre-equilibrated with PBS and washed sequentially with 15 mL elution buffer (PBS containing 0.35 M NaCl and 0.2% Triton X-100) and 15 mL PBS. Finally, elution was performed with 5 mL of 0.1 M glycine buffer (pH = 2.5), and the eluate was immediately neutralized with 1 M Tris-HCl (pH = 8.0) to a pH of approximately 7.4. The collected ADC sample was concentrated using an Amicon ultrafiltration centrifuge tube and the buffer was exchanged with PBS. The resulting product was stored at 4°C in the dark until further use, ultimately yielding the ADC product (Y101S).

[0050] Example 4: ADC effectively reverses TGF-β×PD-L1BsAb resistance

[0051] Multiple tumor-bearing mouse models were constructed to evaluate the in vivo anti-tumor effects of ADC drugs. The EMT-6 tumor-bearing mouse model was described above. In addition, 4T1 and CT26 tumor-bearing mouse models were established using 6-8 week old female BALB / c mice as described below. 6 4T1 breast cancer cells were inoculated into the right mammary fat pad. One week after inoculation, the tumor was about 100 mm. 3 ; 1×10 6 CT26 colorectal cancer cells were inoculated in the right groin. One week after inoculation, the tumor was about 100 mm. 3 Each model was then randomly divided into three groups and intraperitoneally injected with hIgG (6.6 mg / kg), YM101 (9 mg / kg), and ADC drug Y101S (9 mg / kg) every other day for a total of three times. The tumor volume (length × width × width × 0.5) was monitored. The tumor volume exceeded 2500 mm 3 The mice were killed at the end of the experiment. The results showed that Y101S was more effective than YM101 and no drug resistance was observed ( Figure 4 A- Figure 4 B).

[0052] Example 5: ADC has good in vivo safety

[0053] After the third administration of the above-mentioned EMT-6 tumor-bearing mice, peripheral blood was collected for blood biochemical index detection to evaluate the damage of the drug to liver function, kidney function, and heart function. The mouse ocular venous blood was collected using a capillary glass tube. After the blood sample was collected, it was centrifuged at 300g for 10 minutes. The supernatant was placed in a 4°C refrigerator for subsequent testing. The biochemical detection platform is the fully automatic biochemical analyzer LWC400 (Shenzhen Lanyun Medical), and the relevant reagent kits are shown in Table 2. The results showed that after multiple administrations, there was no significant change in the liver function, kidney function, and heart function related indicators of the mice ( Figure 5 ), generally speaking, the safety is good.

[0054] Table 2 Mouse blood biochemical test kit information

[0055]

[0056] Example 6: ADC effectively increases the infiltration of CXCR6+ T cells in the tumor microenvironment

[0057] After EMT-6 tumor-bearing mice received three drug treatments, fresh mouse tumor tissue was collected and single-cell suspensions were prepared for flow cytometry analysis of the number of CXCR6+ T cells in the tumor tissue. The specific flow cytometry protocol is as described above. The results showed that ADC treatment significantly increased the number of CXCL16+ macrophages and dendritic cells infiltrating the tumor ( Figure 6 A). Correspondingly, the number of CXCR6+T cells also increased significantly ( Figure 6 B).

[0058] The above are merely preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be construed as limiting the present invention, and the scope of protection of the present invention should be determined by the scope defined in the claims. Persons skilled in the art will appreciate that improvements and modifications may be made without departing from the spirit and scope of the present invention, and such improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate, characterized in that: The structure of the antibody-drug conjugate is shown in formula (I): Ab-[L-D] n (I) Wherein, Ab is the bispecific antibody YM101 that binds to PD-L1 and TGF-β; L is a cleavable linker; D is a STING agonist; n is the number of Ds connected to Ab via the cleavable linker L, and n is a positive integer; "-" represents a chemical bond.

2. The PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 1, characterized in that: The STING agonists include MSA-2.

3. A method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 1, characterized in that: The steps include: (1) Deprotecting the carrier molecule containing the cleavable linker unit to obtain intermediate I; (2) coupling the intermediate I with a bifunctional linker containing a maleimide group in the presence of an organic base to obtain the intermediate II; (3) condensing the STING agonist and intermediate II in the presence of a condensing agent and a catalyst to form a cleavable linker-drug complex; (4) sulfhydryl modification of the anti-PD-L1 and TGF-β bispecific antibody YM101; (5) performing a coupling reaction between the cleavable linker-drug complex obtained in step (3) and the thiol-ylated antibody obtained in step (4); (6) The target ADC product was obtained by affinity chromatography purification.

4. The method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 3, characterized in that: In step (1), the carrier molecule includes Fmoc-Val-Cit-PAB.

5. The method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 3, characterized in that: In step (2), the bifunctional linker includes NHS-PEG4-Mal, and the molar ratio of the intermediate I to the bifunctional linker is (0.9-1):

1.

6. The method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 3, characterized in that: In step (3), the molar ratio of the STING agonist to the intermediate II is (0.9-1):

1.

7. The method for preparing a PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 3, characterized in that: In step (4), the thiol-forming reagent includes Traut's reagent.

8. The method for preparing the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 3, characterized in that: In step (5), the molar ratio of the cleavable linker-drug complex to the thiolated antibody is 4:

1.

9. Use of the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 1 in the preparation of an anti-tumor drug.

10. Use of the PD-L1 / TGF-β / STING triple-targeted antibody-drug conjugate according to claim 9 in the preparation of anti-tumor drugs, characterized in that: The drug is used to increase the number of CXCL16+ macrophages, dendritic cells and CXCR6+ T cells infiltrating the tumor.

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