Conjugate, preparation method, pharmaceutical composition and application

Abstract

The invention discloses a conjugate, a preparation method, a pharmaceutical composition and application. The conjugate is an immune checkpoint inhibitor coupled with chitosan oligosaccharide. Through the selective binding capacity of the immune checkpoint inhibitor, pediatric immune cells in tumor tissues can be precisely targeted, so that precise targeting of key antigen presenting cells in a tumor immune microenvironment is realized, and the efficiency of immunotherapy is effectively improved. The conjugate can enhance the curative effect of an immune checkpoint inhibitor and remarkably improve the anti-tumor treatment effect by enhancing the antigen presentation and immune cell reaction capacity, meanwhile, the toxic effect of non-targeted tissue can be effectively reduced through in-vivo targeted delivery of the conjugate, and the anti-tumor treatment effect is improved. Compared with the traditional treatment means, the compound has lower side effects and better safety, and has good clinical application potential in the field of tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to tumor immunotherapy, and more particularly to a conjugate, a preparation method, a pharmaceutical composition, and its application. Background Technology

[0002] Tumor immunotherapy is an important direction in cancer treatment. Among them, immune checkpoint inhibitors, represented by antibodies against programmed death receptor-1 (PD-1) and its ligand (PD-L1), have achieved significant efficacy in various cancers by relieving the suppression of immune cells by tumors. However, this type of therapy has limited effectiveness against "cold tumors," that is, tumors with insufficient immune cell infiltration, and some patients develop primary or adaptive resistance. Therefore, how to transform "cold tumors" into "hot tumors" that are sensitive to immunotherapy has become a current research focus.

[0003] The STING pathway is a core component of innate immunity. When abnormal DNA appears in the cytoplasm, the cGAS enzyme catalyzes the generation of a second messenger, activating the STING protein. This, in turn, triggers the production of type I interferon and pro-inflammatory cytokines, promotes dendritic cell maturation and antigen presentation, and ultimately activates the anti-tumor T-cell immune response. Therefore, the STING pathway is considered a key target for reshaping the tumor immune microenvironment and overcoming resistance to immune checkpoint inhibitors.

[0004] However, the clinical translation of STING agonists faces multiple technical bottlenecks. First, some STING agonist molecules suffer from poor in vivo stability, are easily degraded by enzymes, and have unsatisfactory pharmacokinetic properties. Second, when administered systemically, the drug is difficult to accumulate efficiently and specifically in tumor tissue, and the dense matrix and abnormal blood vessels of the tumor can hinder drug penetration. At the same time, exposure to non-target tissues may also trigger severe systemic inflammatory responses, resulting in a narrow therapeutic window.

[0005] Among natural products, chitosan, derived from chitin, is a positively charged natural basic polysaccharide that has been widely studied in the biomedical field due to its good biocompatibility and biodegradability, and is often used to construct drug delivery carriers such as hydrogels. Its degradation product, chitosan oligosaccharide, has better water solubility and is more easily absorbed. Studies have shown that chitosan oligosaccharides with different degrees of polymerization play different roles in drug delivery; for example, low-polymerization monomers may affect the activity of intestinal drug efflux proteins. However, it should be noted that although some studies suggest that certain chitosan derivatives may have immunomodulatory potential, there is no clear evidence to confirm that the low molecular weight degradation product of chitosan, chitosan oligosaccharide, can directly and effectively activate the STING pathway. Summary of the Invention

[0006] Objectives of the invention: The first objective is to provide a conjugate that can be used for tumor immunotherapy; the second objective is to provide a method for preparing the conjugate; and the third objective is to provide a pharmaceutical composition containing the conjugate and its application.

[0007] Technical solution: The conjugate described in this invention is an immune checkpoint inhibitor conjugated with chitosan oligosaccharide.

[0008] Preferably, the molecular weight of the chitosan oligosaccharide is in the range of 3000~5000 Da.

[0009] Preferably, the immune checkpoint inhibitor is a protein-based immune checkpoint inhibitor or a peptide-based immune checkpoint inhibitor.

[0010] Preferably, the immune checkpoint inhibitor is any one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIGIT antibody.

[0011] Preferably, the coupling form is any one of acylation coupling, electrophilic addition coupling, glycosyl site modification coupling, or click chemical reaction coupling.

[0012] The preparation method of the coupling compound of the present invention includes the following steps: (1) Using chitosan oligosaccharide, 4-maleiminobutyric acid-N-succinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as raw materials, 4-maleiminobutyric acid modified chitosan oligosaccharide was obtained by amidation reaction. (2) Using the maleimide-modified chitosan oligosaccharide obtained in step 1 and the immune checkpoint inhibitor as raw materials, a conjugate was obtained through an electrophilic addition reaction.

[0013] Preferably, step 1 includes: (11) Mix equal volumes of chitosan oligosaccharide solution with a concentration of 80-120 mg / mL, 4-maleimide butyric acid-N-succinimide ester dispersion with a concentration of 6.5-8.5 mg / mL, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution with a concentration of 6.5-8.5 mg / mL, and N-hydroxysuccinimide solution with a concentration of 6.5-8.5 mg / mL. Stir and react at room temperature for 12-36 h under nitrogen protection. (12) The reaction product obtained in step 11 was dialyzed and then freeze-dried to obtain 4-maleimide butyric acid modified chitosan oligosaccharide.

[0014] More preferably, the solvents for the chitosan oligosaccharide solution, the 4-maleimide butyric acid-N-succinimide ester dispersion, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution, and the N-hydroxysuccinimide solution are all water.

[0015] Preferably, step 2 includes: (21) Mix equal volumes of 4-maleimide butyric acid modified chitosan oligosaccharide solution with concentration of 80-120 mg / mL and immune checkpoint inhibitor with concentration of 4-6 mg / mL, and stir at room temperature for 12-36 h under nitrogen protection. (22) The reaction product obtained in step 21 was dialyzed and then freeze-dried to obtain the coupling compound.

[0016] Further preferably, the solvent for both the 4-maleimide butyric acid-modified chitosan oligosaccharide solution and the immune checkpoint inhibitor solution is water.

[0017] More preferably, the immune checkpoint inhibitor solution is a mouse-derived anti-PD-L1 antibody solution.

[0018] The pharmaceutical composition of the present invention comprises the aforementioned conjugate as an active ingredient and pharmaceutically acceptable excipients.

[0019] The application of the conjugates or pharmaceutical compositions described in this invention in the preparation of antitumor drugs.

[0020] Preferably, the tumors include, but are not limited to, lung cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, biliary tract cancer, pancreatic cancer, colorectal cancer, renal cell carcinoma, urothelial carcinoma, prostate cancer, endometrial cancer, cervical cancer, ovarian cancer, head and neck squamous cell carcinoma, nasopharyngeal carcinoma, breast cancer, malignant melanoma, squamous cell carcinoma of the skin, Merkel cell carcinoma, mesothelioma, soft tissue sarcoma, primary mediastinal large B-cell lymphoma, and Hodgkin lymphoma.

[0021] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. This conjugate, through the selective binding ability of immune checkpoint inhibitors, can precisely target immune cells in tumor tissues, thereby achieving precise targeting of key antigen-presenting cells in the tumor immune microenvironment and effectively improving the efficiency of immunotherapy. 2. This conjugate enhances the ability of antigen presentation and immune cell responses, including promoting the maturation of dendritic cells (DCs) in the tumor microenvironment and increasing CD8+. + The proportion of T cells enhances the efficacy of immune checkpoint inhibitors and significantly improves the effect of anti-tumor therapy. 3. The targeted delivery of this conjugate in vivo can effectively reduce the toxicity to non-targeted tissues. Compared with traditional treatment methods, it has lower side effects and better safety, and has good clinical application potential in the field of tumor immunotherapy. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the chemical structure of the conjugate obtained in Example 1; Figure 2 This is a flowchart of the synthesis of the conjugate obtained in Example 1; Figure 3 The NMR spectrum of chitosan oligosaccharide; Figure 4 The NMR spectrum of the synthesized 4-maleimide butyric acid-modified chitosan oligosaccharide; Figure 5 The NMR spectrum of the conjugate obtained in Example 1; Figure 6 Fourier transform infrared spectra of chitosan oligosaccharide, 4-maleimide butyric acid-modified chitosan oligosaccharide, and the conjugate obtained in Example 1; Figure 7 Non-reducing polyacrylamide gel electrophoresis images of chitosan oligosaccharide, anti-PD-L1 antibody, and conjugates obtained in Example 1; Figure 8 The image shows the reduced polyacrylamide gel electrophoresis results of the anti-PD-L1 antibody and the conjugate obtained in Example 1. Figure 9 The matrix-assisted laser desorption / ionization tandem time-of-flight mass spectra of the anti-PD-L1 antibody and the conjugate obtained in Example 1; Figure 10 The image shows the anti-tumor results of CT26 subcutaneous tumor model mice injected with the conjugate obtained in Example 1. In the image, a is the tumor growth curve, b is the survival curve, and c is the body weight change curve. Figure 11 The image shows the results of immune cell analysis in the tumor tissue of CT26 subcutaneous tumor model mice on day 18 after intratumoral injection of the conjugate obtained in Example 1. In the image, a is a flow cytometry plot (left) and its quantitative plot (right) of dendritic cell maturation, and b is CD8... + T cell flow cytometry (left) and its quantitative plot (right); Figure 12 The images show in vivo imaging results of CT26 subcutaneous tumor model mice after intratumoral injection of the conjugate obtained in Example 1 labeled with Cy5.5, where a is a fluorescence image and b is a quantitative statistical graph. Figure 13 The image shows the blood routine analysis results of mice with CT26 subcutaneous tumor model treated with intratumoral injection of the conjugate obtained in Example 1 labeled with Cy5.5 on day 50. In the figure, a is the red blood cell count, b is the white blood cell count, c is the hemoglobin concentration, d is the platelet count, and e is the mean corpuscular volume. Figure 14 The image shows the blood biochemical analysis results of mice with CT26 subcutaneous tumor model treated with intratumoral injection of the conjugate obtained in Example 1 labeled with Cy5.5 on day 50. In the figure, a is the result of alanine aminotransferase, b is the result of aspartate aminotransferase, c is the result of blood urea nitrogen, and d is the result of creatinine. Figure 15 H&E staining of major organs in mice with the CT26 subcutaneous tumor model obtained from Example 1, which was injected intratumorally with Cy5.5-labeled conjugate, on day 50 after treatment. Detailed Implementation

[0023] The technical solution of the present invention will be further described below.

[0024] Example 1: Preparation and Characterization of Couplings 1. Preparation of coupling compounds (1) Weigh 200 mg of chitosan oligosaccharide (COS, purchased from Shanghai Dibai Biotechnology Co., Ltd., catalog number P312015), 15 mg of 4-maleimide butyric acid-N-succinimide ester (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number N159716), 15 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E106172), and 15 mg of N-hydroxysuccinimide (Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number H109330), and dissolve or disperse them separately in 2 mL of ultrapure water. Then mix them and react at 800 rpm at 25 °C for 24 h under nitrogen protection. The resulting reaction product was dialyzed in water for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da, with the water changed every 6 h. After freezing at -80℃, it was dried in a vacuum of -80℃ and 20 Pa for 48 h to obtain 4-maleiminobutyric acid modified chitosan oligosaccharide (COS-Mal). (2) Weigh 200 mg of 4-maleimide butyric acid modified chitosan oligosaccharide and 10 mg of... InVivo MAb anti-mousePD-L1 (B7-H1) (aPD-L1, purchased from Bio X cell, catalog number BE0101) were each dissolved separately in 2 mL of ultrapure water, then mixed, and reacted at 25 °C and 800 rpm for 24 h under nitrogen protection. The resulting reaction product was dialyzed in water for 48 h using a dialysis bag with a molecular weight cutoff of 10,000 Da, with the water changed every 6 h. After freezing at -80 °C, it was dried in a vacuum of -80 °C and 20 Pa for 48 h to obtain the conjugate (COS-anti-PD-L1, i.e., COS-aPD-L1), the chemical structure of which is shown in the schematic diagram below. Figure 1 As shown.

[0025] The preparation process of the coupling agent in this embodiment is as follows: Figure 2 As shown.

[0026] 2. Characterization of intermediate products and coupling compounds 2.1 Nuclear Magnetic Resonance Spectroscopy Analysis 10 mg of chitosan oligosaccharide, 4-maleimide butyric acid-modified chitosan oligosaccharide and conjugate were weighed out and dissolved in 0.6 mL of deuterated water by sonication. The solutions were then loaded into NMR tubes and detected using a nuclear magnetic resonance spectrometer (Brook 400 MHz, AVANCE NEO400M).

[0027] Chitosan oligosaccharide NMR spectrum as shown Figure 3 As shown, the NMR spectrum of 4-maleimide butyric acid-modified chitosan oligosaccharide is as follows. Figure 4 As shown, the NMR spectrum of the coupling is as follows: Figure 5 As shown, the peaks with chemical shifts between 2-3 ppm and 4-5 ppm originate from functional groups on the aPD-L1 antibody, indicating that aPD-L1 is coupled to COS.

[0028] 2.2 Fourier Transform Infrared Spectroscopy Analysis Two mg of chitosan oligosaccharide, 4-maleimide butyric acid-modified chitosan oligosaccharide, and conjugate were weighed and placed on the sample stage of a total reflectance infrared spectrometer (Brook, VERTEX 70+HYPERION 2000) for detection.

[0029] Fourier transform infrared spectrum as follows Figure 6 As shown, compared with COS-Mal, COS-aPD-L1 at 1652 cm⁻¹ -1 The disappearance of the small peak at the wavelength indicates that most of the alkenyl groups in COS-Mal participated in the reaction with the aPD-L1 antibody.

[0030] 2.3 Polyacrylamide gel electrophoresis analysis Polyacrylamide gel electrophoresis analysis was performed using a 6% separating gel and a 4% stacking gel.

[0031] After mixing chitosan oligosaccharide, aPD-L1 and the conjugate with loading buffer that does not contain dithiothreitol (DTT), the samples were carefully loaded onto the gel.

[0032] Electrophoresis was performed at a constant voltage of 80 V for 2 hours. The gel after electrophoresis was then immersed in Coomassie Brilliant Blue R-250 staining solution and destained on a shaker at room temperature for 30 minutes. The staining solution was discarded, and a destaining solution containing 30% methanol and 10% glacial acetic acid was added. Destaining was performed on a shaker, changing the destaining solution during the process, until the background was transparent and the bands were clear.

[0033] After decolorization, the gel is transferred to pure water to collect images.

[0034] For polyacrylamide gel electrophoresis with added reducing agent, after mixing aPD-L1 or the conjugate with loading buffer containing DTT, heat at 95°C for 10 min to denature the protein, and then proceed with the same steps as before.

[0035] The results of polyacrylamide gel electrophoresis of samples without the addition of reducing agent are as follows: Figure 7 As shown, compared with the small molecule free COS that does not show a band and the narrower and more concentrated aPD-L1, the band of COS-aPD-L1 is also around 150 kD and shows a tailing feature, which is a characteristic of glycosyl modification. This result indicates that COS was successfully coupled to aPD-L1.

[0036] The results of polyacrylamide gel electrophoresis with added reducing agent in the sample are as follows: Figure 8 As shown, the experimental results once again observed the tailing feature of glycosyl modification, further demonstrating the successful coupling between COS and aPD-L1.

[0037] 2.4 Matrix-assisted laser desorption / ionization tandem time-of-flight mass spectrometry Take aPD-L1 and the conjugate, and use BeyoDesalt. TM Desalting was performed using a G-25 Mini desalting column to prepare a 2 mg / mL solution. This solution was then mixed with acetonitrile containing 10 mg / mL sinapic acid and 0.1% trifluoroacetic acid at a volume ratio of 1:1. The mixture was spotted onto a MALDI TOF target plate, with each sample being 2.5 μL. The plate was then vacuum dried for 4 h and detected using a matrix-assisted laser desorption / ionization tandem time-of-flight mass spectrometer (Bruker, Ultraflextreme MALDITOF / TOF).

[0038] Matrix-assisted laser desorption / ionization tandem time-of-flight mass spectra are as follows: Figure 9 As shown, compared with the raw material aPD-L1, the molecular weight of the main peak of the conjugate COS-aPD-L1 is slightly increased and exhibits a wider morphology. This indicates that COS was successfully conjugated to aPD-L1, causing a change in the molecular weight of the antibody, and the conjugate product may be composed of a mixture of different conjugation ratios.

[0039] Example 2: The conjugate obtained in Example 1 was used to treat CT26 subcutaneous tumor model mice. 1. Monitoring of body weight change curves, tumor growth curves, and survival curves Female BALB / c mice aged 6-8 weeks and weighing 18-22 g were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. CT26 colon cancer cells were subcutaneously inoculated into the right abdomen of the mice at a dose of 500,000 cells per mouse to create tumors.

[0040] When the tumor volume reaches approximately 70 mm 3Tumor-bearing mice were randomly divided into 5 groups: PBS group (PBS), chitosan oligosaccharide group (COS), anti-PD-L1 antibody group (aPD-L1), chitosan oligosaccharide and anti-PD-L1 antibody mixture group (COS+aPD-L1), and conjugate group (COS-aPD-L1), with 16 mice in each group. Among them, 5 mice were used for tumor growth curve monitoring, 6 mice were used for survival curve monitoring, and 5 mice were used for tumor immune cell analysis.

[0041] For mice in the PBS group, 50 μL of sterile PBS was injected intratumorally. For mice in the chitosan oligosaccharide group, 0.5 mg / kg COS was injected intratumorally; For mice in the anti-PD-L1 antibody group, 10 mg / kg aPD-L1 was injected intratumorally; For mice in the chitosan oligosaccharide and anti-PD-L1 antibody mixture group, intratumoral injection of 0.5 mg / kg COS and 10 mg / kg PD-L1 was performed. For mice in the conjugate group, 11 mg / kg COS-aPD-L1 was injected intratumorally. In this experiment, the mouse subcutaneous tumor model was successfully established on the 8th day after inoculation. Therefore, the first administration was recorded as the 8th day. The drug was administered once every 3 days for a total of 4 administrations.

[0042] Starting from the start of drug administration, the body weight of mice in each group was measured every 2 days.

[0043] For mice used for tumor growth curve monitoring, tumor volume was measured every 2 days, and the length and width of the tumor were recorded. The tumor volume was then calculated using the following formula and statistically analyzed: Tumor volume (mm²) 3 = length × width × width ÷ 2.

[0044] For mice used for survival curve monitoring, the tumor volume was defined as 1500 mm. 3 The endpoint of the experiment was reached. Once reached, the mice were euthanized, and their survival time was recorded.

[0045] The tumor growth curve of the CT26 subcutaneous tumor model mouse is as follows: Figure 10 As shown in Figure a, the results indicate that COS-aPD-L1 significantly inhibited tumor growth; the survival curve is shown in Figure a. Figure 10 As shown in b, the results indicate that COS-aPD-L1 significantly prolonged the survival of tumor-bearing mice; the curve is shown in... Figure 10 As shown in c, the results showed that COS-aPD-L1 had no significant effect on the body weight of tumor-bearing mice.

[0046] 2. Analysis of immune cells in tumor tissue The mice used for the aforementioned intratumoral immune cell analysis had tumor tissue collected on day 18 post-treatment. The tissue was ground into a single-cell dispersion suspension, filtered through a 70 μm cell filter, and washed once with PBS containing 5% BSA. Every 10... 6 Cells were treated with 1 μg of TruStain fcX at a concentration of 0.5 mg / mL. TM The Fc receptor blocker (purchased from Biolegend, catalog number 101319) was incubated on ice for 10 min in a 100 μL system.

[0047] For dendritic cell analysis, CD45-APC-Cy7 (catalog number 103116), CD11b-BV421 (catalog number 117330), CD80-PE (catalog number 600056), and CD86-PE-Cy7 (catalog number 105014) were used; for CD8 + T-cell analysis was performed using CD45-APC-Cy7 (catalog number 103116), CD3-PerCP (catalog number 100288), CD4-BV421 (catalog number 100438), and CD8-PE-Cy7 (catalog number 140416); all of these flow cytometry antibodies were purchased from Biolegend, Inc., USA.

[0048] Cells were incubated with flow cytometry antibodies for 1 h and washed once with PBS containing 5% BSA before being analyzed using a BD FACS AriaIII flow cytometer.

[0049] The maturation status of dendritic cells and CD8+ in the tumor tissue of CT26 subcutaneous tumor model mice after COS-aPD-L1 treatment. + The ratio of T cells, for example Figure 11 As shown, the results indicate that COS-aPD-L1 can effectively promote the maturation of dendritic cells in tumor tissue and increase the proportion of tumor-killing CD8+ T cells, suggesting that the conjugate can enhance anti-tumor immunity.

[0050] 3. In vivo distribution assessment Female BALB / c mice, aged 6-8 weeks and weighing 18-22 g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. CT26 colon cancer cells were subcutaneously inoculated into the right ventral region of the mice at a dose of 500,000 cells per mouse to induce tumor formation. Tumors were induced when the tumor volume reached approximately 100 mm². 3 At that time, the intratumoral injection dose of Cy5.5-labeled conjugate was 11 mg / kg, administered only once; in vivo imaging was performed using PerkinElmer IVIS Lumina Series III at 0, 6, 12, 24, 48, 72, and 96 h after administration.

[0051] The preparation method of Cy5.5-labeled conjugate (Cy5.5-COS-aPD-L1) is as follows: 10 mg of COS-aPD-L1, 5 mg of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 5 mg of N-hydroxysuccinimide were weighed and dissolved in 5 mL of ultrapure water and stirred at 800 rpm at room temperature. 2 mg of Cy5.5-COOH (purchased from Shanghai Yi'en Chemical Technology Co., Ltd., catalog number R050967) was weighed and dissolved in 0.1 mL of DMSO and added to the aforementioned reaction solution under stirring. The reaction was carried out for 48 h. The reaction solution was dialyzed in water for 48 h using a dialysis bag with a molecular weight cutoff of 1000 Da, with the water changed every 6 hours. After freezing at -80℃, the solution was dried in a vacuum at -80℃ and 20 Pa for 48 h to obtain the Cy5.5-labeled conjugate.

[0052] The results are as follows Figure 12 As shown, the results indicate that COS-aPD-L1 has a relatively long retention time in mouse tumor tissue.

[0053] 4. Toxic and side effect assessment Female BALB / c mice, aged 6-8 weeks and weighing 18-22 g, were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd. CT26 colon cancer cells were subcutaneously inoculated into the right ventral region of the mice at a dose of 500,000 cells per mouse to induce tumor formation. Tumors were induced when the tumor volume reached approximately 70 mm². 3 On day 8 after subcutaneous tumor inoculation, mice were injected intratumorally with a Cy5.5-labeled conjugate at a dose of 11 mg / kg every 3 days for a total of 4 administrations. On day 50 after subcutaneous tumor inoculation (day 42 after the first administration), blood was collected from the orbital sinus of each mouse. The blood was divided into two portions: one portion was treated with an anticoagulant for routine blood count analysis; the other portion was incubated at room temperature for 30 min, followed by centrifugation at 4000 rpm for 10 min, and the serum was collected for blood biochemical analysis. Simultaneously, blood was collected from healthy female BALB / c mice aged 13-15 weeks and processed in the same manner.

[0054] For whole blood samples, analysis was performed using the Mindray Animal Blood Cell Analyzer BC-2800Vet.

[0055] For serum samples, the following kits were used for detection and analysis: Nanjing Jiancheng Biotechnology Alanine Aminotransferase Microplate Method Kit (Catalog No. C009-2-1), Aspartate Aminotransferase Microplate Method Kit (Catalog No. C010-2-1), Blood Urea Nitrogen Test Kit (Catalog No. C013-2-1), and Creatinine Assay Kit (Catalog No. C011-2-1).

[0056] The results are as follows Figure 13 , 14 As shown, COS-aPD-L1 treatment has no obvious toxic side effects.

[0057] Mice were euthanized after blood collection, and their hearts, livers, spleens, lungs, and kidneys were collected. The tissues were fixed in 4% paraformaldehyde solution for 48 hours, dehydrated in a gradient manner, embedded in paraffin, and cut into 5 μm sections. After dewaxing and hydration, the sections were stained with hematoxylin and eosin (H&E), mounted with neutral resin, and observed and images were acquired under a microscope.

[0058] The results are as follows Figure 15 As shown, COS-aPD-L1 treatment had no significant toxic side effects on the major organs of mice.

Claims

1. A coupling agent, characterized in that, The conjugate is an immune checkpoint inhibitor conjugated with chitosan oligosaccharide.

2. The coupling according to claim 1, characterized in that, The molecular weight range of the chitosan oligosaccharide is 3000~5000 Da.

3. The coupling according to claim 1, characterized in that, The immune checkpoint inhibitor is a protein-based immune checkpoint inhibitor or a peptide-based immune checkpoint inhibitor.

4. The coupling according to claim 3, characterized in that, The immune checkpoint inhibitor is any one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, or anti-TIGIT antibody.

5. The coupling according to claim 1, characterized in that, The coupling form is any one of acylation coupling, electrophilic addition coupling, glycosyl site modification coupling, or click chemical reaction coupling.

6. A method for preparing the coupling compound according to claim 1, characterized in that the step... include: (1) Using chitosan oligosaccharide, 4-maleiminobutyric acid-N-succinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide as raw materials, 4-maleiminobutyric acid modified chitosan oligosaccharide was obtained by amidation reaction. (2) Using the maleimide-modified chitosan oligosaccharide obtained in step 1 and the immune checkpoint inhibitor as raw materials, a conjugate was obtained through an electrophilic addition reaction.

7. The method for preparing the coupling compound according to claim 6, characterized in that, Step 1 includes: (11) Mix equal volumes of chitosan oligosaccharide solution with a concentration of 80-120 mg / mL, 4-maleimide butyric acid-N-succinimide ester dispersion with a concentration of 6.5-8.5 mg / mL, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride solution with a concentration of 6.5-8.5 mg / mL, and N-hydroxysuccinimide solution with a concentration of 6.5-8.5 mg / mL. Stir and react at room temperature for 12-36 h under nitrogen protection. (12) The reaction product obtained in step 11 was dialyzed and then freeze-dried to obtain 4-maleimide butyric acid modified chitosan oligosaccharide.

8. The method for preparing the coupling compound according to claim 6, characterized in that, Step 2 includes: (21) Mix equal volumes of 4-maleiminobutyric acid modified chitosan oligosaccharide solution with concentration of 80-120 mg / mL and immune checkpoint inhibitor with concentration of 4-6 mg / mL, and stir at room temperature for 12-36 h under nitrogen protection. (22) The reaction product obtained in step 21 was dialyzed and then freeze-dried to obtain the coupling compound.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the conjugate of any one of claims 1 to 5 as the active ingredient, and pharmaceutically acceptable excipients.

10. The use of a conjugate according to any one of claims 1 to 5 or a pharmaceutical composition according to claim 9 in the preparation of an antitumor drug.

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