CN117653719A - Tumor in-situ vaccine and preparation method and application thereof - Google Patents

Abstract

The invention discloses a tumor in-situ vaccine, a preparation method and application thereof, and belongs to the technical field of biological medicine. The tumor in-situ vaccine of the invention is prepared from the following raw materials: chemical drugs, polyethylene glycol (PEG), and immunoadjuvants; the chemical drug is modified with bio-orthogonal functional groups, the polyethylene glycol is modified with corresponding bio-orthogonal functional groups, and the bio-orthogonal functional groups of the chemical drug react with the corresponding bio-orthogonal functional groups of the polyethylene glycol to realize cross-linking. The tumor in-situ vaccine provided by the invention is based on the bio-orthogonal hydrogel, the immunogenic chemotherapeutic drug and PEG are subjected to bio-orthogonal reaction to construct a hydrogel system, and the immunoadjuvant is loaded in the hydrogel, so that the tumor recurrence in a primary focus after operation can be effectively inhibited, and the individual in-situ immunotherapy can be realized by utilizing the epitope information of tumor tissues.

Description

A kind of tumor in situ vaccine and its preparation method and application

Technical field

The invention belongs to the field of biomedicine technology, and specifically relates to a tumor in-situ vaccine based on bioorthogonal hydrogel and its preparation method and application.

Background technique

Surgical intervention is usually the first choice for clinical tumor treatment, followed by postoperative chemotherapy, radiotherapy, targeted therapy, hormone therapy, immunotherapy and other treatments, which can effectively prolong patient survival. However, surgery cannot achieve complete tumor removal, especially for solid tumors showing infiltrative growth, which often leads to postoperative in situ recurrence.

As an emerging treatment method, tumor immunotherapy aims to activate the body's own immune system to kill tumors. Treatments to improve the host's tumor immune response include adoptive cells, cytokines, immune checkpoint regulators, tumor vaccines, etc. Among them, tumor vaccines inoculate tumor antigens into patients in the form of cell fragments, proteins or peptides, nucleic acids, etc., thereby overcoming immune suppression, enhancing immunogenicity, and inducing immune responses to eliminate tumors. The U.S. Food and Drug Administration has approved several tumor vaccines, including SμrVaxM. Usually tumor vaccines consist of the above-mentioned tumor antigens and immune adjuvants. Immune adjuvants, also known as immunomodulators or immunopotentiators, are a type of substance that can stimulate and enhance the depth and breadth of antigen immune responses. They themselves have no antigenicity. While improving the antigen adaptive immune response, they can compensate for tumor antigens. Weak immunogenicity, reducing immune tolerance and improving tumor vaccine efficacy. An ideal adjuvant should not only enhance the immune response, but also enable the body to obtain optimal protective immunity after vaccination. In situ vaccines directly utilize tumor antigens produced by immunogenic cell death, including a full set of individual antigen active epitopes. Under the action of immune adjuvants, the body's immune mobilization is achieved. Its mechanism of action includes immune initiation-immune effect-tumor cell death- Antigen release-immune re-initiation-immune re-effect has universal, cyclic and dynamic characteristics. By establishing the body's immune surveillance, it can effectively resist tumor recurrence. Immunogenic chemotherapy drugs are a class of chemical drugs that can induce immunogenic cell death and are characterized by the regulation of damage-related molecular patterns, including the release of adenosine triphosphate in the "find me" signal and the membrane transduction of calreticulin in the "eat me" signal. position, secretion of activation signal high-mobility group proteins, translocation of immune-stimulating signal heat shock protein, etc. Under the joint action of these signaling molecules, innate immunity is activated, recognizes tumor antigens produced by dead cells, and cross-presents them to T cells through antigen-presenting cells to initiate the tumor immune circuit.

Hydrogel is a type of polymer system composed of hydrophilic polymers that are chemically or physically cross-linked and have a three-dimensional network structure. The physical and chemical properties of hydrogel are similar to those of extracellular matrix. It can be used as tissue engineering scaffolds, drug sustained-release systems, medical dressings, etc., and is widely used in the field of biomedicine. The materials used to prepare hydrogels mainly include natural and synthetic polymer systems. The former includes natural products such as proteins and polysaccharides, most of which are biodegradable; the latter includes copolymers such as polyethylene glycol (PEG) and polyacrylic acid. It has a clear structure, controllable molecular weight, no immunogenicity and no antigenicity. PEG is an ethylene oxide polymer with a molecular weight ranging from several hundred to several thousand. According to the number of end groups, it can be divided into mono/bifunctional single-arm polyethylene glycol and multifunctional multi-arm polyethylene glycol. . Some hydrogels cross-linked by PEG have also been approved for marketing, such as HypolTM for wound dressings, OctecTM for controlled drug release, etc. Bioorthogonal reactions refer to reactions that can be carried out in living cells or tissues without interfering with the biochemical processes of organisms. They were first proposed by Carolyn R. Bertozzi in 2003 and have now become a hot spot in the cross-field of biochemistry. Hydrogel systems based on bioorthogonal reactions can form arbitrary shapes that completely match irregular defects, satisfy precise spatiotemporal control, and are ideal drug delivery systems.

Contents of the invention

The purpose of the present invention is to provide a tumor in situ vaccine based on bioorthogonal hydrogel and its preparation method and application.

In order to achieve the above objects, the present invention adopts the following technical solutions:

A tumor in situ vaccine, the preparation raw materials include: chemical drugs, polyethylene glycol (PEG) and immune adjuvants;

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

In the present invention, the combination of the bioorthogonal functional group and the corresponding bioorthogonal functional group is selected from the group consisting of azide and triphenylphosphine, azide and eight-membered cyclic alkynes, tetrazine and trans-cyclooctene, copper-catalyzed azide With terminal alkynes, palladium catalyzed boronic acid and iodobenzene, photocatalyzed alkenes and tetrazole, aldehydes/ketones and amines, thiols and bornene. The combination of different bioorthogonal functional groups has an important impact on the reaction rate and thus the gelation rate.

In the present invention, the chemical drugs mainly refer to chemotherapy drugs that can cause immunogenicity, and are selected from the group consisting of oxaliplatin, mitoxantrone, doxorubicin, epirubicin, idarubicin, and bortezomib. , exemestane, letrozole, irinotecan, bleomycin, methotrexate, nimustine, docetaxel, 5-fluorouracil, or cyclophosphamide. The hydrophilicity and hydrophobicity of chemical drugs can be adjusted through chemical modification.

In the present 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. PEG having 4 arms and a weight average molecular weight of 20,000 is preferred. The hydrophilicity and hydrophobicity of polyethylene glycol are mainly adjusted by molecular weight.

In the present invention, immune adjuvants include various carriers such as inorganic salt adjuvants such as aluminum adjuvants, emulsion adjuvants such as MF59, water-soluble adjuvants such as saponins, particulate antigen presentation system adjuvants such as AS01; CpG-ODN , TLRs, NLRs, CLRs, RLRs, DNA/RNA receptors and other pattern recognition receptor agonists; interleukins, interferons, tumor necrosis factor superfamily, colony-stimulating factors, chemokines, growth factors and other cytokines; CTLA -4, PD-1/PD-L1, OX40 and other immune checkpoint modulators, etc. αOX40 is the only checkpoint regulator discovered so far that can break peripheral immune tolerance and plays an important role in mobilizing and activating immune cells.

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

Step 1: Modify bioorthogonal functional groups with chemical drugs to obtain phase A;

Step 2: Modify the corresponding bioorthogonal functional groups with polyethylene glycol to obtain phase B;

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

Step 4: Before use, mix the above two-phase solution evenly to complete in-situ cross-linking to obtain an in-situ tumor vaccine.

In a preferred embodiment, phase A is a PBS aqueous solution of chemical drugs, and phase B is a PBS aqueous solution of PEG, with a pH of 7.0-8.0, preferably 7.4. After the two phases are mixed, the tumor vaccine for use under intraoperative conditions should solidify rapidly within 5 minutes. In the chemical coupling process of metal ions and photocatalysis, additional processing methods of cross-linking agents and light are required, which is of greater significance for achieving precise spatiotemporal control of bioorthogonal hydrogel systems.

Application of the above-mentioned tumor in situ vaccine in the preparation of postoperative therapeutic drugs for tumors.

The three-dimensional network structure of the bioorthogonal hydrogel system provided by the present invention can be accurately adjusted according to the properties of chemical drugs and PEG, achieving moderate gel rate and gel concentration, avoiding affecting intraoperative operations, and improving mechanical properties and drug release behavior. control to avoid affecting the postoperative efficacy.

The tumor in situ vaccine treatment concept proposed by the present invention aims to use immunogenic chemotherapy drugs to kill residual tumor cells, directly and locally generate a complete set of individual antigenic active epitopes, and complete the construction of individualized tumor vaccines with the assistance of immune adjuvants. Achieve precise treatment and establish immune surveillance of the body to inhibit postoperative tumor recurrence.

The bioorthogonal hydrogel system provided by the present invention can be used under intraoperative conditions, and can also be used as gel spray inhalation, wound dressing filling, external gel coating, subcutaneous implant injection, etc., in which the combination of bioorthogonal functional groups and the properties of the monomer need to be adjusted according to the actual application.

The chemical drug bioorthogonal functional group modification technology provided by the invention can be coupled with proteins and polypeptides and developed as antibody drug conjugates.

Compared with the prior art, the beneficial effects of the present invention are:

As a kind of tumor immunotherapy, the in-situ tumor vaccine based on bioorthogonal hydrogel provided by the present invention can effectively inhibit the recurrence of postoperative tumors in the primary tumor. Immunogenic chemotherapy drugs and PEG construct a hydrogel system through bioorthogonal reactions. The immune adjuvant is loaded in the hydrogel and uses the antigenic epitope information of tumor tissue to achieve personalized in-situ immunotherapy.

Description of drawings

Figure 1 shows the synthesis route of the bioorthogonal hydrogel-based tumor in situ vaccine in Example 1.

Figure 2 is a photo of a double syringe forming a tumor in situ vaccine based on bioorthogonal hydrogel in Example 1.

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

Figure 4 shows the progression of primary tumor in mice during postoperative treatment of the orthotopic glioma model in Example 8.

Figure 5 shows the body weight changes of mice during postoperative treatment of the glioma orthotopic model in Example 8.

Figure 6 is a pathological analysis of the primary tumor in mice after postoperative treatment of the glioma orthotopic model in Example 8.

Detailed ways

The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but should not be understood as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the method, steps or conditions of the present invention shall fall within the scope of the present invention. Experimental methods without specifying specific conditions and reagents without specifying formulas in the examples are all based on conventional conditions in this field.

Example 1

Step 1: Modification of amino bioorthogonal functional groups by oxaliplatin

Place oxaliplatin (OXP, 50mmol) in a 100mL round-bottomed flask, add 30% H ₂ O ₂ (30mL) solution, place it in a 55°C oil bath to react for 2 hours, and continue the reaction at room temperature for 30 minutes before the solution becomes clear. , placed at 4°C overnight, filtered, washed with water, ethanol and ether in sequence, and dried under vacuum overnight.

Place the above product and succinic anhydride (100mmol) in a 100mL round-bottomed flask, add dry N,N-dimethylformamide (DMF, 10mL), place it in a 55°C oil bath to react for 2 hours, and continue the reaction at room temperature overnight. Afterwards, the solution is clarified, spin DMF to dryness, add a small amount of methanol to disperse, settle with a large amount of cold ether, filter, repeat 2-5 times, and vacuum dry overnight.

Place the above product and ethylenediamine (100mmol) in a 100mL round-bottomed flask, add dry DMF (10mL), and react in a 55°C oil bath for 2 hours. Continue the reaction at room temperature overnight. After the solution becomes clear, spin dry the DMF and add Disperse a small amount of methanol, settle with a large amount of cold ether, filter, repeat 2-5 times, and vacuum dry overnight to obtain white powder. Use PBS (1×) with pH 7.4 to prepare a 100 mg/mL aqueous solution as phase A.

Step 2: PEG modified o-phthalaldehyde bioorthogonal functional groups

Place 3,4-dimethylbenzoic acid (100mmol) and N-bromosuccinimide (300mmol) in a 500mL round-bottomed flask, add warm carbon tetrachloride (CCl ₄ , 50mL), and then add Benzoyl peroxide (100 mmol) was placed in an 81°C oil bath and refluxed for 15 hours. Filter the white precipitate while hot, wash with toluene and diethyl ether in sequence, evaporate the filtrate, dissolve the residue in 10% Na ₂ CO ₃ solution, wash with dichloromethane (DCM), acidify, and extract with ethyl acetate to obtain 3,4-bis( Dibromomethyl)benzoic acid.

Place the above product in a 500 mL round-bottomed flask, add 10% Na ₂ CO ₃ (20 mL) solution, and place it in a 70°C oil bath to react for 4 hours. After acidification, extract with acetate, and the product is dissolved in anhydrous methanol, Sc ( OTf) ₃ (100mmol) was treated overnight, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (120mmol) and N-hydroxysuccinimide (120mmol) were added at room temperature. Stir overnight and perform silica gel column purification to obtain 1,3-dimethoxy-1,3-dihydroisobenzofuran-5-carboxylic acid n-succinimide ester.

The above product, 4-arm amino polyethylene glycol (4PEG2000-NH ₂ ) (20 mmol) and pyridine (20 mL) were placed in a 100 mL round-bottomed flask, stirred at room temperature for 48 h, settled with a large amount of cold ether, and filtered. After the intermediate product was dissolved in water, trifluoroacetic acid was added, stirred at room temperature for 1 hour, dialyzed with water for 48 hours, and freeze-dried to obtain a white solid. Use PBS (1×) with pH 7.4 to prepare a 100 mg/mL aqueous solution (PGel) as phase B.

Step 3: Disperse the immune adjuvant αOX40 in the phase A amino-modified oxaliplatin aqueous solution so that the final concentration of αOX40 is 30% (v/v).

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

The synthetic route from step 1 to step 4 above is shown in Figure 1.

After filling the above two phases A and B through a double syringe, inject them at a molar ratio of 2:1. as shown in picture 2. After the two phases A and B come into contact, they can quickly solidify to form a gel within 5 minutes, which is fixed at the bottom of the vial.

Example 2

Step 1: Modify azide bioorthogonal functional groups with doxorubicin

Doxorubicin (Dox, 100mmol) was placed in a 100mL round-bottomed flask, and dry DMF (10mL) was added, followed by 4-azidobutyric acid succinimidyl ester (100mmol) and N,N-diiso Propylethylamine (DIEA, 100 mol) was reacted at room temperature for 24 hours, then purified by liquid phase preparation. The required fractions were freeze-dried and prepared into a 100 mg/mL aqueous solution using PBS (1×) with pH 7.4 as phase A.

Step 2: PEG modified diphenylphosphine orthogonal functional groups

Place 4PEG2000-NH ₂ (20mmol) in a 100mL round-bottomed flask, add dry DCM (10mL), and then add a small amount of triethylamine. After reacting at room temperature for 30 minutes, add 3-diphenylphosphine-4-methoxy Carbonyl benzoate succinimide ester (100 mmol) was continued to react at room temperature for 24 hours, and 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.

Step 3: Disperse the immune adjuvant CpG in the phase A azide-modified doxorubicin aqueous solution so that the final concentration of CpG is 30% (v/v).

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

Example 3

Step 1: Modify azide bioorthogonal functional groups with doxorubicin

Dox (100mmol) was placed in a 100mL round-bottomed flask, dry DMF (10mL) was added, and then 4-azidobutyric acid succinimidyl ester (100mmol) and DIEA (100mmol) were added. After reacting at room temperature for 24 hours, the solution was prepared Phase purification, freeze-dry the required fractions, and prepare a 100 mg/mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

Step 2: PEG modified alkynyl bioorthogonal functional groups

4PEG2000-NH ₂ (20 mmol) was placed in a 100 mL round bottom flask, dry DCM (10 mL) was added, followed by 3-(3-dimethylamino)-1-ethylcarbodiimide hydrochloride (EDC ) activated acetylenic carboxylic acid (100mmol) and N-hydroxysuccinimide (NHS, 100mmol), reacted at room temperature for 24 hours, and then dialyzed and purified to obtain a white solid, which was prepared with PBS (1×) at pH 7.4 to 100 mg/mL. aqueous solution as phase B.

Step 3: Disperse the immune adjuvant PolyI:C in the phase A azide-modified doxorubicin aqueous solution so that the final concentration of PolyI:C is 30% (v/v).

Step 4: Take the above-mentioned phase A and phase B solutions and mix them evenly at a molar ratio of 4:1, add a small amount of copper ions for catalysis, complete the in-situ coupling, and obtain an in-situ tumor vaccine based on bio-orthogonal hydrogel.

Example 4

Step 1: Modification of trans-cycloalkene bioorthogonal functional groups with doxorubicin

Dox (100mmol) was placed in a 100mL round-bottomed flask, dry DMF (10mL) was added, and then EDC-activated azabenzocyclooctanoic acid (100mmol) and NHS (100mmol) were added. After reacting at room temperature for 24 hours, liquid phase purification was performed. , freeze-dry the required fractions, and prepare a 100 mg/mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

Step 2: PEG modified tetrazine orthogonal functional groups

4PEG2000-NH ₂ (20 mmol) was placed in a 100 mL round bottom flask, dry DCM (10 mL) was added, followed by EDC-activated 5-[4-(1,2,4,5-tetrazin-3-yl) Benzyl]-5-oxopentanoic acid (100mmol) and NHS (100mmol) were reacted at room temperature for 24 hours, then dialyzed and purified to obtain a white solid, which was prepared into a 100mg/mL aqueous solution with PBS (1×) at pH 7.4, as B Mutually.

Step 3: Disperse the immune adjuvant lipid A in the aqueous solution of doxorubicin modified with reverse cyclic olefin A, so that the final concentration of lipid A is 30% (v/v).

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

Example 5

Step 1: Modification of alkenyl bioorthogonal functional groups with doxorubicin

Place Dox (100mmol) in a 100mL round-bottomed flask, add dry DMF (10mL), then add EDC-activated 3-butenoic acid (100mmol) and NHS (100mmol), react at room temperature for 24h, and prepare liquid phase purification. The required fractions were freeze-dried and prepared into a 100 mg/mL aqueous solution using PBS (1×) with pH 7.4 as phase A.

Step 2: PEG modified tetrazole bioorthogonal functional groups

4PEG2000-NH ₂ (20 mmol) was placed in a 100 mL round bottom flask, dry DCM (10 mL) was added, followed by EDC-activated 4-(5-phenyl-2H-tetrazol-2-yl)-benzoic acid ( 100mmol) and NHS (100mmol), reacted at room temperature for 24 hours, and then dialyzed and purified to obtain a white solid, which was prepared into a 100mg/mL aqueous solution with PBS (1×) at pH 7.4 as phase B.

Step 3: Disperse the immune adjuvant Cyclic GMP-AMP (cGAMP) in the phase A alkenyl-modified doxorubicin aqueous solution so that the final concentration of Cyclic GMP-AMP (cGAMP) is 30% (v/v).

Step 4: Take the above-mentioned phase A and phase B solutions and mix them evenly at a molar ratio of 4:1. Under ultraviolet light irradiation, complete in-situ coupling to obtain an in-situ tumor vaccine based on bio-orthogonal hydrogel.

Example 6

Step 1: Modify thiol bioorthogonal functional groups with doxorubicin

Place Dox (100mmol) into a 100mL round-bottomed flask, add dry methanol (10mL), then add 2-iminothiolane (100mmol) and a small amount of triethylamine (10mmol), and react under nitrogen at room temperature to avoid light. After 48 hours, wash with diethyl ether, and prepare a 100 mg/mL aqueous solution with PBS (1×) at pH 7.4 as phase A.

Step 2: PEG modified acetyl mercapto bioorthogonal functional group

Place 4PEG2000-NH ₂ (20mmol) in a 100mL round-bottomed flask, add dry DCM (10mL), then add EDC-activated S-acetylthioglycolic acid (100mmol) and NHS (100mmol), react at room temperature for 24h, and then purify by dialysis , a white solid was obtained, which was prepared into a 100 mg/mL aqueous solution using PBS (1×) with pH 7.4 as phase B.

Step 3: Disperse the immune adjuvant lipopolysaccharide in the phase A thiol-modified doxorubicin aqueous solution so that the final concentration of lipopolysaccharide is 30% (v/v).

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

Example 7

In vitro cytotoxicity testing of bioorthogonal hydrogel-based tumor in situ vaccines

Mouse glioma cells GL261 (5000 cells/well) were inoculated into a 96-well plate and incubated in a 37°C 5% CO ₂ incubator for 24 hours. The physical mixture (Mix) of the in situ vaccine (αOX40@PGel), gel component (PGel), oxaliplatin (OXP) and PEG in Example 1 was used with DMEM complete medium or dithiothreitol (DTT) ) were diluted with DMEM complete culture medium and incubated with GL261 cells for 48 hours respectively, followed by washing three times with PBS, adding 100 μL of the cytotoxic reagent Alarmblue per well, and incubating for 2 hours in a 37°C 5% _CO2 incubator. The microplate reader has an excitation wavelength of 540-560nm and an emission wavelength of 590nm to measure the fluorescence value. Use the formula: (fluorescence value of experimental group - fluorescence value of blank group)/fluorescence value of blank group to calculate the cell survival rate in different groups and use Graphpad Prism 9 calculates IC50 and the results are shown in Figure 3. Mix, PGel and αOX40@PGel were used in DMEM complete medium with or without 10mM DTT. The Mix group showed certain cytotoxicity, while neither PGel nor αOX40@PGel showed tumor cell killing effect. After adding 10mM DTT, αOX40@PGel reacted with DTT and the drug was released, thus showing the strongest tumor killing effect among the three groups.

Example 8

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

A homogeneous orthotopic glioma model was established. With the help of a brain stereotaxic instrument, a hole was drilled in the skull of C57BL/6J mice after anesthesia. Luciferase-expressing GL261-Luc cells (100,000cell/5μL) were injected with 0.5μL/ Inoculate the predetermined brain area at an injection speed of 1 min (the bregma is the base point, 1 mm forward, 2 mm to the right, and 2 mm downward). The wound is sutured after stopping the injection for 5 minutes to establish the brain glial of GL261-Luc2-C57BL/6J mice. Tumor orthotopic model. ~10 days later, the model mice were reopened and the tumors were surgically removed under a stereoscope. The original tumor site was filled with an in situ vaccine and the wound was sutured. A small animal in vivo optical three-dimensional imaging system was used to detect and monitor the fluorescence intensity and distribution. Intracranial tumor progression, the results are shown in Figure 4. In Example 1, the mice in the PBS, Mix and PGel groups showed higher fluorescence luminescence intensity, which increased with time; the mice in the αOX40@PGel group had the lowest fluorescence intensity, and did not increase with time. Calculate the tumor volume and weigh the mice, and the results are shown in Figure 5. 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 during the test period and increased slightly in the later period. The mice were sacrificed at scheduled times, and brain tissues were dissected and collected for pathological analysis. The results are shown in Figure 6. The mice in the PBS, Mix and PGel groups all had large tumors in the left brain area, but no obvious tumors were found in the left brain area of the mice in the αOX40@PGel group.

Claims (6)

1. A tumor in situ vaccine, characterized in that: the preparation raw materials include: chemical drugs, polyethylene glycol and immune adjuvants; The chemical drug is modified with bioorthogonal functional groups, the polyethylene glycol is modified with corresponding bioorthogonal functional groups, and the bioorthogonal functional groups of the chemical drug react with the corresponding bioorthogonal functional groups of polyethylene glycol to achieve mutual cross-linking; The combination of the bioorthogonal functional group and the corresponding bioorthogonal functional group is selected from the group consisting of azide and triphenylphosphine, azide and eight-membered cyclic alkynes, tetrazine and trans-cyclooctene, copper-catalyzed azide and terminal alkynes, and palladium Catalytic boronic acid and iodobenzene, photocatalytic alkenes and tetrazole, aldehydes/ketones and amines, thiols and bornene. 2. The tumor in situ vaccine according to claim 1, characterized in that: the chemical drug is selected from the group consisting of oxaliplatin, mitoxantrone, doxorubicin, epirubicin, idarubicin, and bortezo m, exemestane, letrozole, irinotecan, bleomycin, methotrexate, nimustine, docetaxel, 5-fluorouracil, or cyclophosphamide. 3. The tumor in situ vaccine according to claim 1, wherein 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. 4. The tumor in situ vaccine according to claim 1, characterized in that: the immune adjuvant is selected from the group consisting of inorganic salt adjuvants, emulsion adjuvants, water-soluble adjuvants, particulate antigen presenting system adjuvants, and model adjuvants. Identify receptor agonists, cytokines, or immune checkpoint modulators. 5. The preparation method of tumor in situ vaccine according to claim 1, characterized in that: comprising the following steps: Step 1: Modify bioorthogonal functional groups with chemical drugs and dissolve them in aqueous solution as phase A; Step 2: Modify the corresponding bioorthogonal functional groups with polyethylene glycol and dissolve them in the aqueous solution as phase B; Step 3: Disperse the immune adjuvant in phase A or phase B; Step 4: Before use, mix the above two-phase solution evenly to complete in-situ cross-linking to obtain an in-situ tumor vaccine. 6. Application of the tumor in situ vaccine according to claim 1 in the preparation of postoperative therapeutic drugs for tumors.