Hydrogel loaded with ccl25 chemokine and anti-pdl1 antibody and applications thereof

Abstract

The application belongs to the field of tumor treatment drugs, and discloses a hydrogel loaded with CCL25 chemokine and anti-PDL1 antibody and application thereof, and specifically discloses a hydrogel, which comprises a gel base and a substance loaded on the gel base, wherein the substance comprises a chemokine and / or an anti-PDL1 antibody. The hydrogel provided by the application can not only recruit T cells to a specific tumor lesion area, enhance the infiltration of T cells in tumor tissue, but also regulate the function of recruited T cells, further regulate the tumor immune microenvironment, and inhibit tumor growth and postoperative recurrence.

Description

Technical Field

[0001] This invention belongs to the field of tumor therapeutic drugs, specifically relating to a hydrogel loaded with CCL25 chemokine and anti-PDL1 antibody and its application. Background Technology

[0002] In recent years, immunotherapies such as immune checkpoint blockade (ICB) antibodies, chimeric antigen receptor (CAR) T cells, and tumor vaccines have made significant clinical progress, making immunotherapy a first-line treatment for many malignant tumors. It is noteworthy that the therapeutic efficacy of these immunotherapies is highly dependent on T cell function. For example, in certain types of tumors, CD8+... + Insufficient T cell infiltration leads to low clinical response rates to ICB antibody therapy. Even when tumor tissue contains adequately infiltrated T cells, dysfunction of these T cells or immune escape from tumor cells can also result in treatment failure. Therefore, regulating the migration, infiltration, and function (cytotoxicity, cytokine release) of effector T cells in tumor tissue may have a positive impact on immunotherapy efficacy and cancer prognosis.

[0003] Recent reports indicate that CCR9 + CD8 + T cell subsets can trigger powerful anti-tumor effects, thus effectively treating tumors. However, under normal circumstances, CCR9... + CD8 + T cells are mainly distributed in the thymus and small intestine, with a very low infiltration rate in tumor tissue. Furthermore, CCR9... + CD8 + T cells also exhibit increased PD-1 expression, allowing tumor cells highly expressing PD-L1 to escape T cell killing through the PD-1 / PD-L1 axis interaction. The PD-1 / PD-L1 interaction inhibits the proliferation, survival, and function of tumor-infiltrating T cells, ultimately leading to apoptosis of tumor-infiltrating T cells and immune escape by tumor cells. Therefore, enhancing CCR9... + CD8 + Increasing T cell infiltration into tumor tissue while enhancing their ability to kill tumor cells can significantly improve the tumor suppression effect. Summary of the Invention

[0004] The first aspect of the present invention is to provide a hydrogel.

[0005] The second objective of this invention is to provide a method for preparing the hydrogel of the first aspect of this invention.

[0006] A third aspect of the present invention is to provide a hydrogel system.

[0007] The fourth aspect of this invention aims to provide the application of the hydrogel of the first aspect of this invention, the preparation method of the second aspect of this invention, and / or the hydrogel system of the third aspect of this invention.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the present invention provides a hydrogel comprising a gel substrate and a substance loaded on the gel substrate, the substance comprising a chemokine and / or an anti-PDL1 antibody.

[0009] Preferably, the anti-PDL1 antibody is an anti-PDL1 antibody modified with adamantane.

[0010] Preferably, the method for preparing the adamantane-modified anti-PDL1 antibody includes the following steps: 1) Carboxylated PEG was modified with 1-adamantane formyl chloride to obtain adamantane-modified polyethylene glycol; 2) Mix adamantane-modified polyethylene glycol with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dimethyl sulfoxide. After the first reaction, add anti-PDL1 antibody and perform a second reaction to obtain adamantane-modified anti-PDL1 antibody.

[0011] Preferably, the conditions for the first reaction are: stirring at 2-6°C for 20-50 min; further, stirring at 2-4°C for 20-40 min; and even further, stirring at 2-4°C for 30-40 min.

[0012] Preferably, the conditions for the second reaction are: stirring at 2-6°C for 10-14 h; further, stirring at 2-4°C for 11-13 h; and even further, stirring at 2-4°C for 11-12 h.

[0013] Preferably, step 1) specifically includes: mixing carboxylated PEG, triethylamine and tetrahydrofuran to obtain solution A; mixing 1-adamantaneformyl chloride with tetrahydrofuran to obtain solution B; slowly adding solution B to solution A, and continuing to add for 3-4 hours to obtain adamantane-modified polyethylene glycol.

[0014] More preferably, the molar ratio of the carboxylated PEG, triethylamine, and 1-adamantaneformyl chloride is 1:1~2:1~2.

[0015] More preferably, the triethylamine is triethylamine that has been dehydrated by reflux of phosphorus pentoxide.

[0016] More preferably, the tetrahydrofuran is tetrahydrofuran that has undergone a dehydration process.

[0017] More preferably, step 1) further includes filtration and ether precipitation steps.

[0018] More preferably, the ether precipitation is performed using anhydrous ether at -20°C.

[0019] Preferably, the mass ratio of the adamantane-modified polyethylene glycol, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and anti-PDL1 antibody in step 2) is 1~2:0.05~0.1:0.05~1:1; more preferably 1.5~2:0.05~0.8:0.05~0.9:1; and even more preferably 1.66:0.06:0.08:1.

[0020] Preferably, the gel substrate is one or more of sodium alginate, carbomer, carboxymethyl chitosan, and cyclodextrin-modified sodium alginate.

[0021] Preferably, the gel substrate is sodium alginate modified with cyclodextrin; more preferably, it is sodium alginate modified with β-cyclodextrin.

[0022] Preferably, the β-cyclodextrin-modified sodium alginate is prepared by esterification of sodium alginate with β-cyclodextrin.

[0023] Preferably, the preparation method of the β-cyclodextrin-modified sodium alginate includes the following steps: dispersing sodium alginate in N,N-dimethylformamide and heating to 40~60℃; adding toluenesulfonic acid and stirring for 20~30 min; adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and β-cyclodextrin and stirring for 20~26 h; washing to obtain β-cyclodextrin-modified sodium alginate.

[0024] More preferably, the mass ratio of sodium alginate, toluenesulfonic acid, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and β-cyclodextrin is 1:1~1.5:0.5~1:0.1~0.5; more preferably, it is 1:1~1.3:0.6~1:0.1~0.2; and even more preferably, it is 1:1.3:0.96:0.1.

[0025] Preferably, the chemokine is one or more of CXC chemokine, CC chemokine, CX3C chemokine, and C chemokine.

[0026] Preferably, the chemokine includes one or more of CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL11, CCL13, CCL14, CCL15, CCL16, CCL17, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL9, CXCL8, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL16, CXCL17, IL8, XCL1, XCL2, and CX3CL1.

[0027] Preferably, the chemokine is a CC chemokine; more preferably, it is CCL25.

[0028] A second aspect of the present invention is to provide a method for preparing the hydrogel of the first aspect of the present invention, comprising the following steps: mixing a gel substrate and a substance loaded on the gel substrate, reacting, thereby obtaining a hydrogel.

[0029] Preferably, the reaction involves reacting a gel substrate and a mixture of substances loaded on the gel substrate in a CaCl2 solution.

[0030] Preferably, the mass ratio of the gel substrate to the substance loaded on the gel substrate is 1:0.00001~0.5; more preferably 1:0.00001~0.4; and even more preferably 1:0.00001~0.3.

[0031] Preferably, the mass concentration ratio of the gel substrate to the CaCl2 is 1:0.1~0.5; further, it is 1:0.2~0.5; and even further, it is 1:0.3~0.5.

[0032] A third aspect of the present invention is to provide a hydrogel system comprising the hydrogel of the first aspect of the present invention and an anti-PDL1 antibody.

[0033] Preferably, the anti-PDL1 antibody is an anti-PDL1 antibody modified with adamantane.

[0034] Preferably, the hydrogel is administered via site-specific (intratumoral) delivery, and the anti-PD1 antibody is administered via intravenous delivery.

[0035] ALG-βCD hydrogel loaded with CCL25 chemokine and aPDL1 antibody CCL25&Ad-aPDL1This system can significantly enhance its anti-tumor effect by modulating the immune microenvironment through multi-level promotion of T cell anti-tumor function via recruitment, fixation, and reactivation. The strategy primarily utilizes βCD-modified sodium alginate (ALG-βCD) hydrogels, which can load the chemokine CCL25 and possess abundant supramolecular binding sites (βCD). The CCL25 chemokine loaded in the hydrogel can recruit CCR9 cells. + CD8 + T cells enter tumor tissue. Simultaneously, βCD in the hydrogel can immobilize the Ad-aPDL1 antibody within the hydrogel. Furthermore, Ad-aPD1 administered via tail vein injection can bind to CCR9. + CD8 + On the surface of T cells, Ad-aPDL1 and Ad-aPD1 are recruited into the tumor tissue in a "hitchhiking" manner and further immobilized in ALG-βCD hydrogel. Then, the immobilized Ad-aPDL1 and Ad-aPD1 promote CD8 activation. + The interaction between T cells and tumor cells, and the restoration of the killing function and activity of CD8+ T cells. Based on this strategy, CCR9 + CD8 + T cells can be effectively recruited into tumor tissues, and their activation and anti-tumor function are significantly promoted, ultimately achieving significant anti-tumor and recurrence-inhibiting effects in orthotopic and postoperative models of mouse melanoma (B16F10). Figure 10 ).

[0036] The fourth aspect of the present invention is to provide the application of the hydrogel of the first aspect of the present invention, the preparation method of the second aspect of the present invention, and / or the hydrogel system of the third aspect of the present invention in any one of (1) to (12); (1) Preparation of anti-tumor products; (2) To prepare products that inhibit tumor recurrence; (3) Enhance the chemotactic capacity of T cells; (4) Prepare products that enhance the chemotactic ability of T cells; (5) Promotes the interaction between T cells and tumor cells; (6) Prepare products that promote the interaction between T cells and tumor cells; (7) Alleviate T cell depletion; (8) Prepare products to alleviate T cell depletion; (9) Regulate the tumor immune microenvironment; (10) Prepare products that regulate the tumor immune microenvironment; (11) Inhibits tumor growth; (12) Prepare products that inhibit tumor growth.

[0037] Preferably, the product includes, but is not limited to, drugs, reagents, and kits.

[0038] Preferably, the T cells include CCR9. + CD8 + T cells.

[0039] The beneficial effects of this invention are: The hydrogel provided by this invention can not only recruit T cells to specific tumor lesion areas and enhance the infiltration of T cells in tumor tissue, but also regulate the function of recruited T cells and further regulate the tumor immune microenvironment, inhibiting tumor growth and postoperative recurrence.

[0040] Furthermore, by promoting the interaction between T cells and tumor cells through supramolecular interaction between adamantane and cyclodextrin, T cell depletion is alleviated, and the killing power of T cells against tumor cells is significantly enhanced.

[0041] The ALG-βCD provided by this invention CCL25&Ad-aPDL1 The / Ad-aPD1 hydrogel system employs a multi-layered strategy to recruit, immobilize, and reactivate T cells in vivo, thereby modulating the immune microenvironment and significantly enhancing anti-tumor efficacy. This system not only eliminates tumors in situ but also reduces postoperative tumor recurrence. This hydrogel system, loaded with CCL25 chemokine and aPDL1 antibody, provides a novel and effective strategy for anti-tumor immunotherapy and holds great promise for clinical translation. Attached Figure Description

[0042] Figure 1 The preparation and characterization results of β-cyclodextrin-modified sodium alginate (ALG-βCD) are shown in Figure a, where a represents the preparation principle of ALG-βCD, b represents the results of infrared spectroscopy analysis, and c represents the results of thermogravimetric analysis.

[0043] Figure 2 For ALG-βCD CCL25&Ad-aPDL1 Transmission electron microscopy (TEM) image of the in-situ hydrogel.

[0044] Figure 3 This is a diagram illustrating the drug release effect of the ALG-βCD hydrogel.

[0045] Figure 4 For ALG-βCD CCL25&Ad-aPDL1 Stability diagram of in-situ hydrogel in different buffer solutions.

[0046] Figure 5 A schematic diagram of the synthesis of Ad-PEG-COOH and its 1H NMR spectrum ( 1 H-NMR spectrum.

[0047] Figure 6The interaction between Ad-aPD1 antibody and β-CD was determined by isothermal titration calorimetry, where a represents the interaction between 1-adamantaneamine and β-CD determined by isothermal titration calorimetry, and b represents the interaction between Ad-aPD1 antibody and β-CD determined by isothermal titration calorimetry.

[0048] Figure 7 For ALG-βCD CCL25 The chemotaxis of T cells by hydrogels in vitro is shown in figure a, b, and c. The figure is a schematic diagram of the experiment, b is a statistical graph of the cell number results in ALG-βCDCCL25 hydrogels containing different concentrations of CCL25, and c is a laser confocal microscope image.

[0049] Figure 8 For ALG-βCD CCL25&Ad-aPDL1 The in vivo antitumor effect of the / Ad-aPD1 hydrogel system is shown in the figures. Figure a represents the experimental process, figure b represents the statistical results of mouse tumor volume, figure c represents the statistical results of mouse tumor weight, and figure d represents the actual image of mouse tumors. represent p <0.001, represent p <0.01.

[0050] Figure 9 For ALG-βCD CCL25&Ad-aPDL1 The Ad-aPD1 hydrogel system's effect on inhibiting tumor recurrence in a postoperative tumor model is illustrated in the figures. Figure a shows the experimental process, figure b shows the statistical results of mouse tumor volume, figure c shows the statistical results of mouse tumor weight, and figure d shows the actual tumor in the mouse. represent p <0.001, represent p <0.005.

[0051] Figure 10 For ALG-βCD CCL25&Ad-aPDL1 / Schematic diagram of the anti-tumor principle of the Ad-aPD1 hydrogel system. Detailed Implementation

[0052] The present invention will now be described in detail with reference to specific embodiments, but this does not limit the scope of the invention.

[0053] Unless otherwise specified, the materials and reagents used in this embodiment are commercially available.

[0054] Example 1: Preparation of β-cyclodextrin-modified sodium alginate (ALG-βCD) In this embodiment, β-cyclodextrin-modified sodium alginate (ALG-βCD) is prepared via an esterification reaction between the carboxyl group on sodium alginate and the hydroxyl group on β-cyclodextrin. The specific preparation method is as follows: 3.0 g of sodium alginate (ALG) was weighed and dispersed in 20 mL of N,N-dimethylformamide (DMF). The solution was heated to 50 °C, and 3.9 g of p-toluenesulfonic acid (PTSA) was added. After stirring for 30 min, 2.9 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.3 g of β-cyclodextrin (βCD) were added, and the reaction was carried out under continuous stirring for 24 h. After the reaction was completed, the product was repeatedly washed with anhydrous ethanol, and the resulting product was lyophilized. The lyophilized product was identified as β-cyclodextrin-modified sodium alginate and named ALG-βCD. Figure 1 (a)

[0055] The obtained ALG-βCD was characterized by infrared spectroscopy and thermogravimetric analysis. The specific method for infrared spectroscopy is as follows: 5 mg of ALG, βCD, and ALG-βCD were weighed and mixed with 0.5 g of potassium bromide, then compressed into tablets. Subsequently, each sample was analyzed using an infrared spectrometer. The results are as follows: Figure 1 As shown in Figure b, ALG-βCD at 1730 cm⁻¹ -1 The presence of a distinct carbonyl stretching vibration absorption peak indicates the successful synthesis of ALG-βCD. The specific thermogravimetric analysis method is as follows: 2 mg of ALG, βCD, and ALG-βCD were weighed and added to the crucible of the thermogravimetric analyzer, and the mass change of each sample was detected and analyzed during the temperature program. The results show that the mass ratio of βCD in the synthesized ALG-βCD is 9.14% (…). Figure 1 (c) indicates the successful synthesis of ALG-βCD.

[0056] Example 2 Synthesis of adamantane-modified antibodies Ad-aPD1 and Ad-aPDL1 (1) Synthesis of Ad-PEG-COOH Adamantane-modified polyethylene glycol (Ad-PEG-COOH) was obtained by modifying carboxylated PEG with 1-adamantaneformyl chloride. The specific method is as follows: HO-PEG113-COOH and triethylamine (triethylamine after dehydration by reflux of phosphorus pentoxide) were dissolved in tetrahydrofuran (THF, tetrahydrofuran after dehydration) to obtain solution A; 1-adamantaneformyl chloride was separately dissolved in THF to obtain solution B. Solution B was slowly added dropwise to solution A, and the reaction was continued at 0℃ for 4 h (the molar ratio of HO-PEG113-COOH, triethylamine, and 1-adamantaneformyl chloride was 1:1.1:1.1). After the reaction was completed, the mixture was filtered, and the resulting product was precipitated with cold diethyl ether (anhydrous diethyl ether at -20℃) to obtain adamantane-modified polyethylene glycol, named Ad-PEG-COOH.

[0057] Weigh 10 mg of the prepared Ad-PEG-COOH, add 0.8 mL of DMSO-d6, dissolve, and then perform 1H NMR spectroscopy. The results are as follows: Figure 5 As shown, the 1H-NMR results indicate the successful synthesis of Ad-PEG-COOH.

[0058] (2) Successful synthesis of adamantane-modified antibodies Ad-aPD1 and Ad-aPDL1 8.3 mg of Ad-PEG-COOH, 0.4 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 0.3 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in dimethyl sulfoxide (DMSO), and the reaction was carried out under stirring for 30 min. Subsequently, 5 mg of anti-PDL1 antibody (aPDL1, Bioxcell, Clone: ​​10F.9G2) or anti-PD1 antibody (aPD1, Bioxcell, Clone: ​​RMP1-14) was added to the above solution, and the reaction was carried out under stirring at 4 °C for 12 h. After the reaction was completed, the above solution was filtered to remove impurities using an ultrafiltration tube to obtain the adamantane-modified antibody, named Ad-aPDL1 or Ad-aPD1.

[0059] The obtained Ad-aPD1 antibody was further characterized, firstly by detecting its ability to interact with cyclodextrin using isothermal titration calorimetry (ITC). Figure 6 As shown, the Ad-aPD1 antibody still retains its good supramolecular interaction with cyclodextrin, indicating the successful modification with adamantane.

[0060] Example 3: ALG-βCD loaded with chemokine CCL25 and Ad-aPDL1 antibody CCL25&Ad-aPDL1 Preparation of in-situ hydrogels ALG-βCDCCL25&Ad-aPDL1 The preparation method of the in-situ hydrogel includes the following steps: 10 mg of ALG-βCD prepared in Example 1 is dissolved in ultrapure water to prepare an ALG-βCD solution with a concentration of 10 mg / mL. 0.2 μg of CCL25 (Biolegend, Cat#, 589306) and 2 mg of Ad-aPDL1 prepared in Example 2 are added to 1 mL of the ALG-βCD solution. After stirring and mixing, the mixture is added to 1 mL of a 20 mM CaCl2 solution. After the reaction, the in-situ hydrogel ALG-βCD is obtained. CCL25&Ad-aPDL1 .

[0061] The prepared ALG-βCD CCL25&Ad-aPDL1 The structure of the in-situ hydrogel was characterized as follows: ALG-βCD CCL25&Ad-aPDL1 The in-situ hydrogel was lyophilized and imaged using scanning electron microscopy. The results showed that ALG-βCD... CCL25&Ad-aPDL1 In-situ hydrogels possess a three-dimensional, loose, and porous structure with pore sizes ranging from 100 to 200 μm. Figure 2 ).

[0062] Example 4 This example is used to investigate the sustained-release properties and stability of the ALG-βCD prepared in Example 1. Details are as follows: A hydrogel was formed by mixing 1 mL of ALG-βCD (10 mg / mL), 1 mL of CaCl2 (20 mM), and 2 mg of BSA (BSA was selected as the model drug for hydrogel drug release). The prepared hydrogel (1 mL) was added to a 10 mL centrifuge tube, followed by 2 mL of PBS. The centrifuge tube was placed in a shaker at 37°C and gently shaken. The supernatant was collected at predetermined time points, and the original PBS was replaced with fresh PBS. The amount of BSA released was then detected using a BCA kit.

[0063] The results are as follows Figure 3 As shown, the hydrogel formed by ALG-βCD and calcium ions exhibits a significant sustained-release effect on bovine albumin (BSA), maintaining good release even after 12 days. Furthermore, its prolonged presence in various buffers (H2O, DMEM, and PBS) also demonstrates the long-term structural stability of the ALG-βCD hydrogel. Figure 4 ).

[0064] Example 5 ALG-βCD CCL25 The chemotactic ability of hydrogels on T cells ALG-βCD CCL25The preparation method of the hydrogel includes the following steps: 10 mg of ALG-βCD prepared in Example 1 is dissolved in ultrapure water to prepare an ALG-βCD solution with a concentration of 10 mg / mL. 0.2 μg of CCL25 is added to 1 mL of the ALG-βCD solution, and after stirring and mixing, it is added to 1 mL of a 20 mM CaCl2 solution. After reaction, the ALG-βCD hydrogel is obtained. CCL25 .

[0065] Further investigation of ALG-βCD with different concentration gradients was conducted using Ibidi μ-Slide Chemotaxis3D. CCL25 The chemotactic effect of hydrogels on T cells in vitro. Specifically, ALG-βCD containing CCL25 (concentrations of 200, 100, 50, and 25 ng / mL) was used. CCL25 Hydrogel and CD8+ T cell suspension (5×10 5 The Ibidi μ-Slide Chemotaxis3D was added to two separate chambers of the hydrogel. A chemokine concentration gradient gradually formed between the hydrogel and the T cells. After incubating the Ibidi μ-Slide Chemotaxis3D at 37°C for 4 h, the CD8+ T cells in the hydrogel were observed and counted using a laser confocal microscope.

[0066] The results are as follows Figure 7 As shown, ALG-βCD CCL25 Hydrogels exhibit strong T cell chemotaxis in vitro, and this chemotactic effect is directly proportional to the concentration of chemokines in the hydrogel.

[0067] Example 6 An ALG-βCD CCL25&Ad-aPDL1 / Ad-aPD1 hydrogel system, including ALG-βCD prepared in Example 4 CCL25&Ad-aPDL1 Hydrogel and Ad-aPD1 prepared in Example 2.

[0068] Example 7 A type of ALG CCL25 / Ad-aPD1 hydrogel system, including ALG CCL25 Hydrogel and Ad-aPD1 prepared in Example 2.

[0069] ALG CCL25 The hydrogel was prepared as follows: 10 mg of ALG was dissolved in ultrapure water to prepare an ALG solution with a concentration of 10 mg / mL. 0.2 μg of CCL25 was added to 1 mL of the ALG solution, and after stirring and mixing, the solution was added to 1 mL of a 20 mM CaCl2 solution. After the reaction, ALG was obtained.CCL25 Hydrogel.

[0070] Example 8 An ALG-βCD CCL25 / Ad-aPD1 hydrogel system, including ALG-βCD prepared in Example 5 CCL25 Hydrogel and Ad-aPD1 prepared in Example 2.

[0071] Example 9 A type of ALG CCL25&Ad-aPDL1 Hydrogel systems, including ALG CCL25&Ad-aPDL1 Hydrogel and Ad-aPD1 prepared in Example 2.

[0072] ALG CCL25&Ad-aPDL1 The hydrogel was prepared as follows: 10 mg of ALG was dissolved in ultrapure water to prepare an ALG solution with a concentration of 10 mg / mL. 0.2 μg of CCL25 and 2 mg of Ad-aPDL1 prepared in Example 2 were added to 1 mL of the ALG solution. After stirring and mixing, the mixture was added to 1 mL of a 20 mM CaCl2 solution. After the reaction, ALG was obtained. CCL25&Ad-aPDL1 Hydrogel.

[0073] Example 10 ALG-βCD CCL25&Ad-aPDL1 / Ad-aPD1 The anti-tumor effect of hydrogel systems in vivo Four-week-old female C57BL / 6J mice were selected, and 2×10⁻⁶ mg / L was injected into the right dorsal side of the mice. 5 A suspension of B16-F10 cells was cultured routinely for 6 days until the tumor volume reached approximately 50 mm. 3 Then, proceed to the next experiment.

[0074] The tumor volume is approximately 50 mm. 3 Mice were randomly divided into 7 groups, with X mice in each group. The 7 groups were G1: PBS, G2: Ad-aPD1, G3: aPD1, and G4: ALG. CCL25 / Ad-aPD1,G5:ALG-βCD CCL25 / Ad-aPD1,G6:ALG CCL25&Ad-aPDL1 / Ad-aPD1,G7:ALG-βCD CCL25&Ad-aPDL1 / Ad-aPD1. PBS (G1) or Ad-aPD1 (5 mg / kg) (G2, G4, G5, G6, and G7) or aPD1 (5 mg / kg) (G3) were administered intravenously, while simultaneously injecting 50 μL of ALG or ALG-βCD solution containing CCL25 (5 μg) and Ad-aPDL1 (100 μg) into the tumor (i.e., no substance was injected in G1, G2, and G3, and ALG was injected in G4).CCL25 Hydrogel, G5 injection of ALG-βCD CCL25 Hydrogel, G6 injection ALG CCL25&Ad-aPDL1 Hydrogel, G7 injection of ALG-βCD CCL25&Ad-aPDL1 (In-situ hydrogel). Tumor volume and body weight were monitored every other day. After 12 days of treatment, the mice were euthanized, and the tumor tissue was removed and the tumor weight was measured.

[0075] The results are as follows Figure 8 As shown, in mice via ALG-βCD CCL25&Ad-aPDL1 After treatment with the / Ad-aPD1 hydrogel system, tumor growth in mice was significantly inhibited compared to other groups, indicating that the in situ hydrogel system significantly improved the anti-tumor effect by chemotaxis, fixation of T cells, and enhancement of their interaction with tumor cells.

[0076] Example 11 ALG-βCD CCL25&Ad-aPDL1 The effect of Ad-aPD1 hydrogel system on inhibiting tumor recurrence in postoperative tumor models Will contain 2×10 5 A suspension of B16-F10 cells was inoculated into the right back of mice. After 10 days of routine feeding, the mice were randomly divided into 7 groups (G1: PBS, G2: Ad-aPD1, G3: aPD1, G4: ALG). CCL25 / Ad-aPD1,G5:ALG-βCD CCL25 / Ad-aPD1,G6:ALG CCL25&Ad-aPDL1 / Ad-aPD1,G7:ALG-βCD CCL25&Ad-aPDL1 / Ad-aPD1), and tumor resection surgery was performed, leaving 1% residual tumor postoperatively to simulate postoperative residue. Mice were then intravenously injected with PBS (G1) or Ad-aPD1 (5 mg / kg) (G2, G4, G5, G6, and G7) or aPD1 (5 mg / kg) (G3). 50 μL of ALG or ALG-βCD solution containing CCL25 (5 μg) and Ad-aPDL1 (100 μg) was injected into the surgical wound (i.e., G1, G2, and G3 received no injection, G4 received ALG). CCL25 Hydrogel, G5 injection of ALG-βCD CCL25 Hydrogel, G6 injection ALG CCL25&Ad-aPDL1 Hydrogel, G7 injection of ALG-βCD CCL25&Ad-aPDL1 (In-situ hydrogel). Tumor volume and weight are monitored every other day.

[0077] like Figure 9 As shown, after ALG-βCD CCL25&Ad-aPDL1The Ad-aPD1 hydrogel system significantly inhibited tumor growth in mice after treatment, while other treatment groups showed significant tumor recurrence. These results indicate that ALG-βCD... CCL25&Ad-aPDL1 The / Ad-aPD1 hydrogel system not only has a significant inhibitory effect on in situ tumors, but also has a very significant inhibitory ability on postoperative tumor recurrence.

[0078] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. A hydrogel comprising a gel substrate and a substance loaded on the gel substrate, the substance comprising a chemokine and an anti-PDL1 antibody, wherein the anti-PDL1 antibody is an anti-PDL1 antibody modified with adamantane, and the gel substrate is sodium alginate modified with cyclodextrin.

2. The hydrogel according to claim 1, characterized in that, The chemokine is one or more of CXC chemokine, CC chemokine, CX3C chemokine, and C chemokine.

3. The hydrogel according to claim 2, characterized in that, The chemokine is a CC chemokine.

4. The hydrogel according to claim 2 or 3, characterized in that, The preparation method of the adamantane-modified anti-PDL1 antibody includes the following steps: 1) Carboxylated PEG was modified with 1-adamantane formyl chloride to obtain adamantane-modified polyethylene glycol; 2) Mix adamantane-modified polyethylene glycol with 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and dimethyl sulfoxide. After the first reaction, add anti-PDL1 antibody and perform a second reaction to obtain adamantane-modified anti-PDL1 antibody.

5. The hydrogel according to claim 4, characterized in that, The conditions for the first reaction described in 2) are stirring at 2~6℃ for 20~50 min; the conditions for the second reaction described in 2) are stirring at 2~6℃ for 10~14 h.

6. A method for preparing the hydrogel according to any one of claims 1 to 5, comprising the following steps: The gel substrate and the substance are mixed and reacted to obtain a hydrogel.

7. The preparation method according to claim 6, characterized in that, The reaction involves reacting a mixture of a gel substrate and the substance in a CaCl2 solution.

8. The preparation method according to claim 7, characterized in that, The mass ratio of the gel substrate to the substance is 1:0.00001~0.

5.

9. The preparation method according to claim 7, characterized in that, The mass concentration ratio of the gel substrate to the CaCl2 is 1:0.1~0.

5.

10. The use of the hydrogel according to any one of claims 1 to 5 or the preparation method according to any one of claims 6 to 9 in any one of (1) to (7); (1) Preparation of anti-tumor products; (2) To prepare products that inhibit tumor recurrence; (3) Prepare products that enhance the chemotactic ability of T cells; (4) Prepare products that promote the interaction between T cells and tumor cells; (5) Prepare products to alleviate T cell depletion; (6) Prepare products that regulate the tumor immune microenvironment; (7) Prepare products that inhibit tumor growth.

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