A tumor in situ vaccine and a preparation method and application thereof

Abstract

The application discloses a tumor in-situ vaccine and a preparation method and application thereof, and belongs to the technical field of biological medicine. The tumor in-situ vaccine is prepared from the following raw materials: a chemical drug, polyethylene glycol (PEG) and an immune adjuvant. The chemical drug is modified with a biological orthogonal functional group, the PEG is modified with a corresponding biological orthogonal functional group, and the biological orthogonal functional group of the chemical drug is reacted with the corresponding biological orthogonal functional group of the PEG to realize mutual crosslinking. The tumor in-situ vaccine provided by the application is based on a biological orthogonal hydrogel, an immunogenic chemotherapy drug and PEG are combined into a hydrogel system through a biological orthogonal reaction, and the immune adjuvant is loaded in the hydrogel. The tumor in-situ vaccine can not only effectively inhibit the recurrence of a postoperative tumor in a primary tumor, but also can utilize antigen epitope information of a tumor tissue to realize individualized in-situ immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an in situ tumor vaccine based on bioorthogonal hydrogels, its preparation method, and its application. Background Technology

[0002] Surgical intervention is usually the first-line treatment for clinical tumors, followed by various therapies such as chemotherapy, radiotherapy, targeted therapy, hormone therapy, and immunotherapy, which can effectively prolong the patient's survival. However, surgery cannot completely remove tumors, especially solid tumors that exhibit invasive growth, often leading to recurrence in situ after surgery.

[0003] As an emerging treatment approach, tumor immunotherapy aims to activate the body's own immune system to kill tumors. Treatments that improve the host's tumor immune response include adoptive cells, cytokines, immune checkpoint modulators, and tumor vaccines. Among these, tumor vaccines deliver tumor antigens to the patient in the form of cellular fragments, proteins or peptides, or nucleic acids, thereby overcoming immunosuppression, enhancing immunogenicity, and inducing an immune response to eliminate tumors. The U.S. Food and Drug Administration has approved several tumor vaccines, including SμrVaxM. Typically, tumor vaccines consist of the aforementioned tumor antigens and immune adjuvants. Immune adjuvants, also known as immunomodulators or immune enhancers, are substances that can stimulate and enhance the depth and breadth of antigen-mediated immune responses. They themselves are not antigenic; while improving antigen-adaptive immune responses, they can compensate for the weak immunogenicity of tumor antigens, reduce immune tolerance, and improve the efficacy of tumor vaccines. An ideal adjuvant should not only enhance the immune response but also ensure optimal protective immunity after vaccination. In situ vaccines directly utilize tumor antigens generated by immunogenic cell death, containing a complete set of individual antigenic active epitopes. Under the action of immune adjuvants, they achieve immune mobilization. Their mechanism of action includes immune initiation-immune effector-tumor cell death-antigen release-immune re-initiation-immune re-effector, exhibiting universality, cyclicality, and dynamic characteristics. By establishing immune surveillance, they can effectively resist tumor recurrence. Immunogenic chemotherapy drugs are a class of chemical drugs that can induce immunogenic cell death. Their characteristic feature is the regulation of damage-related molecular patterns, including the release of adenosine triphosphate (ATP) as a "find me" signal, the membrane translocation of calreticulin as a "eat me" signal, the secretion of high-mobility group box proteins as activation signals, and the translocation of heat shock proteins as immune stimulation signals. Under the combined action of these signaling molecules, innate immunity is activated, recognizing tumor antigens generated by dying cells and initiating the tumor immune circuit by cross-presenting antigens to T cells via antigen-presenting cells.

[0004] Hydrogels are a class of polymeric systems with a three-dimensional network structure, composed of hydrophilic polymers cross-linked chemically or physically. The physicochemical properties of hydrogels are similar to those of the extracellular matrix, making them widely used in the biomedical field as tissue engineering scaffolds, drug delivery systems, and medical dressings. Materials used to prepare hydrogels mainly include natural and synthetic polymeric systems. Natural polymers include proteins, polysaccharides, and other natural products, most of which are biodegradable; synthetic polymers include copolymers such as polyethylene glycol (PEG) and polyacrylic acid, which have well-defined structures, controllable molecular weights, and are non-immunogenic and non-antigenic. PEG is an ethylene oxide polymer with molecular weights ranging from several hundred to several thousand. Based on the number of end groups, it can be divided into monofunctional / bifunctional single-arm PEG and multifunctional multi-arm PEG. Some PEG-crosslinked hydrogels have been approved for marketing, such as Hypol™ for wound dressings and Octec™ for controlled drug release. Bioorthogonal reactions are reactions that can occur in living cells or tissues without interfering with the organism's own biochemical processes. First proposed by Carolyn R. Bertozzi in 2003, they have become a hot topic in the interdisciplinary field of biochemistry. Hydrogel systems based on bioorthogonal reactions can form arbitrary shapes that perfectly match irregular defects, allowing for precise spatiotemporal control and making them ideal drug delivery systems. Summary of the Invention

[0005] The purpose of this invention is to provide an in situ tumor vaccine based on bioorthogonal hydrogels, its preparation method, and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] An in situ tumor vaccine, the raw materials for its preparation include: chemical drugs, polyethylene glycol (PEG) and immune adjuvants;

[0008] The chemical drug is modified with bioorthogonal functional groups, and the polyethylene glycol is modified with corresponding bioorthogonal functional groups. The bioorthogonal functional groups react with each other to achieve cross-linking.

[0009] In this invention, the combination of the bioorthogonal functional group and its corresponding bioorthogonal functional group is selected from azide and triphenylphosphine, azide and eight-membered cycloalkyne, tetrazine and trans-cyclooctene, copper-catalyzed azide and terminal alkyne, palladium-catalyzed boric acid and iodobenzene, photocatalyzed olefin and tetrazolium, aldehyde / ketone and amine, and mercapto and borneolene. Different combinations of bioorthogonal functional groups have a significant impact on the reaction rate, and thus affect the gelation rate.

[0010]

[0011] In this invention, the chemical drugs mainly refer to chemotherapeutic drugs capable of inducing immunogenicity, selected from oxaliplatin, mitoxantrone, doxorubicin, epirubicin, idarubicin, bortezomib, exemestane, letrozole, irinotecan, bleomycin, methotrexate, nimustine, docetaxel, 5-fluorouracil, or cyclophosphamide. The hydrophilicity or hydrophobicity of the chemical drugs can be adjusted through chemical modification.

[0012]

[0013] In this invention, the polyethylene glycol is a single-arm polyethylene glycol or a multi-arm polyethylene glycol with a weight-average molecular weight between 200 and 20,000. Preferably, it is a PEG with four arms and a weight-average molecular weight of 20,000. The hydrophilicity and hydrophobicity of polyethylene glycol are mainly adjusted by its molecular weight.

[0014]

[0015] In this invention, immune adjuvants include various carriers such as inorganic salt adjuvants like aluminum adjuvants, emulsion adjuvants like MF59, water-soluble adjuvants like saponins, and microparticle antigen presentation system adjuvants like ASO1; pattern recognition receptor agonists such as CpG-ODN, TLRs, NLRs, CLRs, RLRs, and DNA / RNA receptors; cytokines such as interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, and growth factors; and immune checkpoint modulators such as CTLA-4, PD-1 / PD-L1, and αOX40. αOX40 is the only checkpoint modulator discovered to date capable of breaking peripheral immune tolerance and plays a crucial role in mobilizing and activating immune cells.

[0016] The preparation method of the above-mentioned tumor in situ vaccine includes the following steps:

[0017] Step 1: Modify biological orthogonal functional groups with chemical drugs to obtain phase A;

[0018] Step 2: Modify polyethylene glycol with corresponding biological orthogonal functional groups to obtain phase B;

[0019] Step 3: Disperse the immune adjuvant in an aqueous solution of phase A or phase B;

[0020] Step 4: Before use, mix the two phase solutions evenly to complete in-situ cross-linking and obtain the tumor in-situ vaccine.

[0021] In a preferred embodiment, phase A is a PBS aqueous solution of the chemical drug, and phase B is a PBS aqueous solution of PEG, with a pH of 7.0-8.0, preferably 7.4. After mixing the two phases, the tumor vaccine to be applied under intraoperative conditions should rapidly solidify within 5 minutes. In the chemical coupling process of metal ions and photocatalysis, additional treatment methods such as cross-linking agents and light irradiation are required, which are of greater significance for achieving precise spatiotemporal control of the bioorthogonal hydrogel system.

[0022] The application of the above-mentioned tumor in situ vaccines in the preparation of postoperative tumor treatment drugs.

[0023] The three-dimensional network structure of the bioorthogonal hydrogel system provided by this invention can be precisely adjusted according to the properties of chemical drugs and PEG, so as to achieve moderate gelation rate and gel concentration, avoid affecting intraoperative operation, and controllable mechanical properties and drug release behavior, thus avoiding affecting postoperative efficacy.

[0024] The tumor in situ vaccine therapy concept proposed in this invention aims to use immunogenic chemotherapy drugs to kill residual tumor cells, directly generate a complete set of individual antigenic active epitopes locally, and complete the construction of individualized tumor vaccines with the assistance of immune adjuvants, thereby achieving precision treatment, establishing the body's immune surveillance, and inhibiting postoperative tumor recurrence.

[0025] The bioorthogonal hydrogel system provided by this invention can be used during surgery, as well as for gel spray inhalation, wound dressing filling, topical gel application, subcutaneous implant injection, etc. The combination of bioorthogonal functional groups and the properties of monomers need to be adjusted according to the actual application.

[0026] The chemical drug bioorthogonal functional group modification technology provided by this invention can be conjugated with proteins and peptides to develop antibody drug conjugates.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] The tumor in situ vaccine based on bioorthogonal hydrogel provided by this invention serves as a tumor immunotherapy, effectively inhibiting postoperative tumor recurrence within the primary lesion. An immunogenic chemotherapeutic drug and PEG are synthesized into a hydrogel system via a bioorthogonal reaction, with an immune adjuvant loaded within the hydrogel. This allows for personalized in situ immunotherapy by utilizing the antigenic epitope information of tumor tissue. Attached Figure Description

[0029] Figure 1 This is the synthetic route for the tumor in situ vaccine based on bioorthogonal hydrogels in Example 1.

[0030] Figure 2 This is a photograph of the tumor in situ vaccine formed by the dual-syringe in Example 1 based on bioorthogonal hydrogel.

[0031] Figure 3 This is an in vitro cytotoxicity test of the tumor in situ vaccine based on bioorthogonal hydrogel in Example 7.

[0032] Figure 4 This describes the progression of the primary tumor in mice during postoperative treatment of the orthotopic glioma model in Example 8.

[0033] Figure 5 The change in body weight of mice during postoperative treatment in the orthotopic glioma model of Example 8.

[0034] Figure 6 This is a pathological analysis of the primary tumor lesion in mice after postoperative treatment in the orthotopic glioma model of Example 8. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0036] Example 1

[0037] Step 1: Oxaliplatin modification of amino bioorthogonal functional groups

[0038] Oxaliplatin (OXP, 50 mmol) was placed in a 100 mL round-bottom flask, and 30% H2O2 (30 mL) solution was added. The mixture was placed in an oil bath at 55 °C for 2 h and reacted at room temperature for 30 min. The solution became clear and was placed at 4 °C overnight. The mixture was filtered, washed successively with water, ethanol and ether, and dried under vacuum overnight.

[0039] The above product and succinic anhydride (100 mmol) were placed in a 100 mL round-bottom flask, and dry N,N-dimethylformamide (DMF, 10 mL) was added. The mixture was placed in an oil bath at 55 °C for 2 h, and the reaction was continued overnight at room temperature. The solution became clear, DMF was evaporated to dryness, a small amount of methanol was added to disperse the solution, and the mixture was precipitated with a large amount of cold diethyl ether. The mixture was filtered, and the reaction was repeated 2-5 times. The mixture was then dried under vacuum overnight.

[0040] The above product was placed in a 100 mL round-bottom flask with ethylenediamine (100 mmol), and 10 mL of dry DMF was added. The mixture was placed in an oil bath at 55 °C for 2 h, and the reaction was continued overnight at room temperature. After the solution became clear, the DMF was evaporated to dryness, a small amount of methanol was added for dispersion, and the mixture was precipitated with a large amount of cold diethyl ether. The mixture was filtered, and the reaction was repeated 2-5 times. The product was then dried under vacuum overnight to obtain a white powder. A 100 mg / mL aqueous solution was prepared using PBS (1×) at pH 7.4 as phase A.

[0041] Step 2: PEG modification of o-phthalaldehyde bioorthogonal functional groups

[0042] 3,4-Dimethylbenzoic acid (100 mmol) and N-bromosuccinimide (300 mmol) were placed in a 500 mL round-bottom flask, and warm carbon tetrachloride (CCl4, 50 mL) was added, followed by benzoyl peroxide (100 mmol). The mixture was refluxed in an oil bath at 81 °C for 15 h. The white precipitate was filtered off while hot, washed successively with toluene and diethyl ether, and the filtrate was evaporated. The residue was dissolved in 10% Na2CO3 solution, washed with dichloromethane (DCM), acidified, and extracted with ethyl acetate to give 3,4-bis(dibromomethyl)benzoic acid.

[0043] The above product was placed in a 500 mL round-bottom flask, and 20 mL of 10% Na2CO3 solution was added. The mixture was reacted in an oil bath at 70 °C for 4 h. After acidification, the product was extracted with acetate. The product was dissolved in anhydrous methanol, treated with 100 mmol of Sc(OTf)3 overnight, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (120 mmol) and N-hydroxysuccinimide (120 mmol) were added. The mixture was stirred overnight at room temperature and purified by silica gel column chromatography to give 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid n-succinimide ester.

[0044] The above product, 4-arm amino polyethylene glycol (4PEG2000-NH2) (20 mmol), and pyridine (20 mL) were placed in a 100 mL round-bottom flask and stirred at room temperature for 48 h. The mixture was then precipitated with a large amount of cold diethyl ether and filtered. The intermediate product was dissolved in water, and trifluoroacetic acid was added. The mixture was stirred at room temperature for 1 h, dialyzed against water for 48 h, and lyophilized to obtain a white solid. A 100 mg / mL aqueous solution (PGel) was prepared using PBS (1×) at pH 7.4 as phase B.

[0045] Step 3: Disperse the immunoadjuvant αOX40 in an aqueous solution of amino-modified oxaliplatin in phase A to achieve a final concentration of 30% (v / v).

[0046] Step 4: Take the above A phase and B phase solutions and mix them evenly at a molar ratio of 2:1 to complete the in situ coupling and obtain the tumor in situ vaccine based on bioorthogonal hydrogel.

[0047] The synthesis routes for steps one through four above are as follows: Figure 1 As shown.

[0048] After filling phases A and B using a dual-syringe syringe, inject them at a molar ratio of 2:1. For example... Figure 2 As shown. After phases A and B come into contact, they can quickly solidify into a gel within 5 minutes, which is then fixed to the bottom of the vial.

[0049] Example 2

[0050] Step 1: Doxorubicin modifies orthogonal functional groups of azide organisms

[0051] Doxorubicin (Dox, 100 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of dry DMF was added. Then, 100 mmol of 4-azidobutyrate succinimide and N,N-diisopropylethylamine (DIEA, 100 mol) were added. After reacting at room temperature for 24 h, liquid phase purification was prepared, and the required fraction was lyophilized and prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

[0052] Step 2: PEG modification of diphenylphosphine orthogonal functional groups

[0053] 4PEG2000-NH2 (20 mmol) was placed in a 100 mL round-bottom flask, and 10 mL of dry DCM was added. Then, a small amount of triethylamine was added. After reacting at room temperature for 30 min, 100 mmol of 3-diphenylphosphino-4-methoxycarbonylbenzoic acid succinimide ester was added, and the reaction was continued at room temperature for 24 h. The mixture was purified by dialysis to obtain a white solid, which was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

[0054] Step 3: Disperse the immune adjuvant CpG in the A-phase azide-modified doxorubicin aqueous solution to achieve a final CpG concentration of 30% (v / v).

[0055] Step 4: Mix the above A phase and B phase solutions at a molar ratio of 4:1 to complete the in situ coupling and obtain the tumor in situ vaccine based on bioorthogonal hydrogel.

[0056] Example 3

[0057] Step 1: Doxorubicin modifies orthogonal functional groups of azide organisms

[0058] Dox (100 mmol) was placed in a 100 mL round-bottom flask, and dry DMF (10 mL) was added. Then 4-azidobutyrate succinimide ester (100 mmol) and DIEA (100 mmol) were added. After reacting at room temperature for 24 h, liquid phase purification was prepared. The required fraction was lyophilized and prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

[0059] Step 2: PEG modification of alkyne-based bioorthogonal functional groups

[0060] 4PEG2000-NH2 (20 mmol) was placed in a 100 mL round-bottom flask, and dried DCM (10 mL) was added. Then, acetylene carboxylic acid (100 mmol) activated by 3-(3-dimethylamino)-1-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS, 100 mmol) were added. After reacting at room temperature for 24 h, the mixture was purified by dialyzing to obtain a white solid. The solid was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

[0061] Step 3: Disperse the immunoadjuvant PolyI:C in the A-phase azide-modified doxorubicin aqueous solution to achieve a final PolyI:C concentration of 30% (v / v).

[0062] Step 4: Mix the above A phase and B phase solutions at a molar ratio of 4:1, add a small amount of copper ions for catalysis, complete the in-situ coupling, and obtain the tumor in-situ vaccine based on bioorthogonal hydrogel.

[0063] Example 4

[0064] Step 1: Doxorubicin modification of trans-cycloene bioorthogonal functional groups

[0065] Dox (100 mmol) was placed in a 100 mL round-bottom flask, and dry DMF (10 mL) was added. Then, EDC-activated azirconium octanoic acid (100 mmol) and NHS (100 mmol) were added. After reacting at room temperature for 24 h, liquid phase purification was prepared. The required fraction was lyophilized and prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

[0066] Step 2: PEG modification of tetrazine orthogonal functional groups

[0067] 4PEG2000-NH2 (20 mmol) was placed in a 100 mL round-bottom flask, and dried DCM (10 mL) was added. Then, EDC-activated 5-[4-(1,2,4,5-tetraazine-3-yl)benzyl]-5-oxovaleric acid (100 mmol) and NHS (100 mmol) were added. After reacting at room temperature for 24 h, the mixture was purified by dialysis to obtain a white solid. The solid was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

[0068] Step 3: Disperse the immune adjuvant lipid A in an aqueous solution of doxorubicin modified with the A-opioid to achieve a final concentration of 30% (v / v).

[0069] Step 4: Mix the above A phase and B phase solutions at a molar ratio of 4:1 to complete the in situ coupling and obtain the tumor in situ vaccine based on bioorthogonal hydrogel.

[0070] Example 5

[0071] Step 1: Doxorubicin modifies alkenyl bioorthogonal functional groups

[0072] Dox (100 mmol) was placed in a 100 mL round-bottom flask, and dried DMF (10 mL) was added. Then, EDC-activated 3-butenoic acid (100 mmol) and NHS (100 mmol) were added. After reacting at room temperature for 24 h, liquid phase purification was prepared. The required fraction was lyophilized and prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

[0073] Step 2: PEG modification of tetrazolium bioorthogonal functional groups

[0074] 4PEG2000-NH2 (20 mmol) was placed in a 100 mL round-bottom flask, and dried DCM (10 mL) was added. Then, EDC-activated 4-(5-phenyl-2H-tetrazol-2-yl)-benzoic acid (100 mmol) and NHS (100 mmol) were added. After reacting at room temperature for 24 h, the mixture was purified by dialysis to obtain a white solid. The solid was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

[0075] Step 3: Disperse the immunoadjuvant Cyclic GMP-AMP (cGAMP) in an aqueous solution of doxorubicin modified with phase A alkenyl to achieve a final concentration of 30% (v / v).

[0076] Step 4: Mix the above A phase and B phase solutions at a molar ratio of 4:1 until homogeneous. Under ultraviolet light irradiation, complete the in-situ coupling to obtain the tumor in-situ vaccine based on bioorthogonal hydrogel.

[0077] Example 6

[0078] Step 1: Doxorubicin modifies thiol-based bioorthogonal functional groups

[0079] Dox (100 mmol) was placed in a 100 mL round-bottom flask, and dry methanol (10 mL) was added. Then 2-iminothiacyclopentane (100 mmol) and a small amount of triethylamine (10 mmol) were added. The mixture was reacted under nitrogen protection at room temperature in the dark for 48 h. After washing with ether, the mixture was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

[0080] Step 2: PEG modification of acetyl thiol bioorthogonal functional groups

[0081] 4PEG2000-NH2 (20 mmol) was placed in a 100 mL round-bottom flask, and dried DCM (10 mL) was added. Then, EDC-activated S-acetyl mercaptoacetic acid (100 mmol) and NHS (100 mmol) were added. After reacting at room temperature for 24 h, the mixture was purified by dialysis to obtain a white solid. The solid was prepared into a 100 mg / mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

[0082] Step 3: Disperse the immune adjuvant lipopolysaccharide in phase A thiol-modified doxorubicin aqueous solution to achieve a final lipopolysaccharide concentration of 30% (v / v).

[0083] Step 4: Mix the above A phase and B phase solutions at a molar ratio of 4:1 to complete the in situ coupling and obtain the tumor in situ vaccine based on bioorthogonal hydrogel.

[0084] Example 7

[0085] In vitro cytotoxicity test of tumor in situ vaccine based on bioorthogonal hydrogel

[0086] Mouse glioma cells GL261 (5000 cells / well) were seeded in 96-well plates and incubated at 37°C for 24 h in a 5% CO2 incubator. The physical mixture of the orthotopic vaccine (αOX40@PGel), gel component (PGel), oxaliplatin (OXP), and PEG from Example 1 was diluted with DMEM complete medium or DMEM complete medium containing dithiothreitol (DTT) and incubated with GL261 cells for 48 h. After washing three times with PBS, 100 μL of Alarm Blue cytotoxicity reagent was added to each well, and the cells were incubated at 37°C for 2 h in a 5% CO2 incubator. Fluorescence values ​​were measured using a microplate reader at an excitation wavelength of 540-560 nm and an emission wavelength of 590 nm. Cell viability was calculated using the formula: (experimental group fluorescence value - blank group fluorescence value) / blank group fluorescence value. IC50 was calculated using Graphpad Prism 9. The results are shown below. Figure 3 As shown, in DMEM complete medium with or without 10 mM DTT, the Mix group (PGel and αOX40@PGel) exhibited some cytotoxicity, while PGel and αOX40@PGel did not show tumor cell killing activity. Upon addition of 10 mM DTT, αOX40@PGel reacted with DTT, releasing the drug and thus exhibiting the strongest tumor-killing activity among the three groups.

[0087] Example 8

[0088] In vivo efficacy of tumor in situ vaccines based on bioorthogonal hydrogels

[0089] A homologous orthotopic glioma model was established. Using a stereotaxic instrument, C57BL / 6J mice were anesthetized and a hole was drilled in the skull. Luciferase-expressing GL261-Luc cells (100,000 cells / 5 μL) were injected at a rate of 0.5 μL / min into predetermined brain regions (1 mm anteriorly, 2 mm to the right, and 2 mm inferiorly, with the anterior fontanelle as the base). After a 5-minute pause, the wound was sutured, establishing the GL261-Luc2-C57BL / 6J mouse orthotopic glioma model. After approximately 10 days, the model mice underwent a second craniotomy, and the tumor was surgically removed under stereoscopic guidance. The primary lesion was then filled with the orthotopic vaccine, and the wound was sutured. A small animal in vivo optical three-dimensional imaging system was used to detect the fluorescence intensity and distribution and monitor intracranial tumor progression. The results are as follows: Figure 4 As shown in the figure. In Example 1, mice in the PBS, Mix, and PGel groups exhibited higher fluorescence intensity, which increased over time; mice in the αOX40@PGel group showed the lowest fluorescence intensity, which did not increase over time. Tumor volume was calculated and mouse weight was measured, and the results are as follows. Figure 5 As shown in the figure, the body weight of mice in the PBS, Mix, and PGel groups decreased to varying degrees over time, while the body weight of mice in the αOX40@PGel group remained stable throughout the experimental period, with a slight increase in the later stages. Mice were sacrificed at predetermined times, and brain tissue was dissected and collected for pathological analysis. The results are as follows. Figure 6 As shown, mice in the PBS, Mix, and PGel groups all had large tumors in the left brain region, while mice in the αOX40@PGel group did not show obvious tumors in the left brain region.

Claims

1. A tumor in situ vaccine, characterized in that, The raw materials for preparation include: chemical drugs, polyethylene glycol, and immune adjuvants; The chemical drug is oxaliplatin, which is modified with amino bioorthogonal functional groups; The polyethylene glycol is a 4-arm amino polyethylene glycol modified with o-phthalaldehyde bioorthogonal functional groups; The amino bioorthogonal functional group reacts with the o-phthalaldehyde bioorthogonal functional group to achieve cross-linking; The immune adjuvant is αOX40; The molar ratio of the chemical drug to polyethylene glycol is 2:

1.

2. The method for preparing the tumor in situ vaccine according to claim 1, characterized in that, Includes the following steps: Step 1: Modify the amino bioorthogonal functional group of the chemical drug oxaliplatin and dissolve it in an aqueous solution to form phase A; Step 2: Polyethylene glycol is modified with the corresponding bio-orthogonal functional groups of o-phthalaldehyde and dissolved in an aqueous solution to form phase B; Step 3: Disperse the immune adjuvant in phase A to achieve a final concentration of 30% v / v; Step 4: Before use, mix phase A and phase B solutions uniformly at a molar ratio of 2:1 to complete in-situ cross-linking and obtain the tumor in-situ vaccine.

3. The use of the tumor in situ vaccine according to claim 1 in the preparation of a drug for inhibiting postoperative recurrence of glioma.

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