Engineered myeloid cells and methods of making and uses thereof

Abstract

The present application relates to the field of vaccines, in particular to an engineered myeloid cell and a preparation method and use thereof. The present application provides an antigen-modified gel, an engineered myeloid cell and use thereof, wherein the antigen-modified gel comprises a plurality of immunostimulants and tumor antigens, attracts monocytes to migrate into and differentiate into antigen-presenting cells, and the activated and antigen-loaded monocytes further migrate out of the gel to lymph nodes, and can efficiently induce an anti-tumor immune response of lymphocytes such as T cells.

Description

Technical Field

[0001] This invention relates to the field of vaccines, specifically to an engineered myeloid cell, its preparation method, and its uses. Background Technology

[0002] Tumor vaccines have long been a hot research topic in the field of tumor immunotherapy. They can activate the body's immune system, induce anti-tumor immune responses, and have good clinical value. To better activate anti-tumor immune responses, current vaccine systems often consist of multiple components, including adjuvants that enhance antigen immunogenicity and antigens at multiple sites. Meanwhile, recent years have seen breakthroughs in tumor vaccines, with the concept of cell vaccines being proposed and its rationale validated. However, current cell vaccines suffer from drawbacks such as high preparation costs and long development cycles, resulting in limited clinical translational value. Therefore, designing new cell vaccine platforms, optimizing these shortcomings, and combining them with existing vaccine systems is crucial. Summary of the Invention

[0003] This invention aims to at least partially solve one of the technical problems in the related art. Therefore, one object of this invention is to provide an antigen-modified gel, engineered myeloid cells, and their uses. The antigen-modified gel contains various immunostimulants and tumor antigens, attracting monocytes to migrate in and causing them to differentiate into antigen-presenting cells. The activated monocytes, loaded with antigens, further migrate out of the gel and travel to lymph nodes, effectively inducing an anti-tumor immune response from lymphocytes such as T cells.

[0004] Therefore, in a first aspect, the present invention provides a modified gel. According to an embodiment of the present invention, the gel comprises:

[0005] Methacrylate-modified hyaluronic acid;

[0006] Crosslinking agent;

[0007] Colony stimulating factors;

[0008] Immunostimulants

[0009] And antigenic peptides,

[0010] The gel contains a porous structure, the colony-stimulating factor and the immunostimulant are encapsulated inside the porous structure, and the antigenic peptide is loaded on the surface of the gel.

[0011] To address the shortcomings of current cell vaccines, the inventors discovered an antigen-modified gel. This gel, with its permeable macroporous structure, effectively encapsulates colony-stimulating factor GM-CSF and other active components such as immunostimulants. Hyaluronic acid modified with methacrylate reduces immunostimulation. A special cross-linking agent connects the antigen to the gel backbone, and thiol-induced polymerization further modifies the gel surface with antigenic peptides. Using an in situ in vivo cell-engineered approach, the gel, containing multiple immunostimulants and tumor antigens, attracts monocytes, causing them to differentiate into antigen-presenting cells. The activated, antigen-loaded monocytes then migrate out of the gel to lymph nodes, efficiently inducing anti-tumor immune responses from T cells and other lymphocytes.

[0012] According to an embodiment of the present invention, the colony stimulating factor is GM-CSF.

[0013] According to an embodiment of the present invention, the immunostimulant includes at least one selected from STING agonists, NLR agonists, and TLR agonists.

[0014] According to an embodiment of the present invention, the NLR agonist comprises NOD2.

[0015] According to an embodiment of the present invention, the TLR agonist includes at least one selected from TLR1 / 2, TLR3, TLR4, TLR7 / 8, and TLR9.

[0016] The inventors discovered that encapsulating the aforementioned specific types of colony-stimulating factors and immunostimulants in a gel can recruit and activate antigen-presenting cells, including dendritic cells and monocytes.

[0017] According to an embodiment of the present invention, the crosslinking agent comprises ammonium persulfate and N,N,N',N'-tetramethyldiethylamine. This enables the antigen to be stably attached to the gel.

[0018] According to an embodiment of the present invention, the gel is obtained by freeze polymerization of a mixture of methacrylate-modified hyaluronic acid, a crosslinking agent, a colony stimulating factor, and an immunostimulating agent.

[0019] According to an embodiment of the present invention, the antigenic peptide is loaded onto the gel surface by the following method:

[0020] (1) The gel obtained by freeze polymerization is reacted with compound A to obtain the first intermediate product;

[0021] (2) Obtain the thiol-modified antigenic peptide, mix the thiol-modified antigenic peptide with a thioctic acid monomer derivative, and perform a polymerization reaction to obtain a second intermediate product;

[0022] (3) Mix the first intermediate product and the second intermediate product to obtain a gel modified with the antigen peptide;

[0023] The structural formula of compound A is as follows:

[0024]

[0025]

[0026] According to an embodiment of the present invention, the lipoic acid monomer derivative is a compound of formula B, and the structural formula of compound B is:

[0027]

[0028] Wherein, R is selected from H, -COO - Or substituents containing a guanidine group.

[0029] According to an embodiment of the present invention, the lipoic acid monomer derivative is selected from at least one of the following:

[0030]

[0031] A second aspect of the present invention provides engineered myeloid cells. According to an embodiment of the present invention, the engineered myeloid cells are linked to an antigenic peptide via a linker, wherein the linker is a polymer formed by the polymerization reaction of a lipoic acid monomer derivative.

[0032] The lipoic acid monomer derivative creates a certain distance between the antigen and the gel surface, which facilitates the reaction with thiol groups on the cell surface and utilizes the connection between the antigen peptide and the cell.

[0033] According to an embodiment of the present invention, the lipoic acid monomer derivative is a compound of formula B, and the structural formula of compound B is:

[0034]

[0035] Wherein, R is selected from H, -COO - Or substituents containing a guanidine group.

[0036] According to an embodiment of the present invention, the lipoic acid monomer derivative is selected from at least one of the following:

[0037]

[0038] A third aspect of the present invention provides a method for preparing engineered myeloid cells. According to an embodiment of the present invention, the method includes:

[0039] Myeloid cells are mixed with the modified gel described in the first aspect, and the thiol groups on the cell membrane surface of the myeloid cells undergo a thiol-disulfide bond exchange reaction with the modified gel, so that the antigenic peptide loaded on the surface of the modified gel is attached to the surface of the myeloid cells or enters the myeloid cells, in order to obtain engineered myeloid cells.

[0040] Currently, most cell vaccine technologies are based on immune cells such as dendritic cells and T cells, while research on the numerous myeloid cells in the body is limited. By mixing the modified gel with myeloid cells and carrying out a series of reactions, engineered myeloid cells with surface-modified antigenic peptides can be obtained, which can be used as cell vaccines for disease prevention.

[0041] According to an embodiment of the present invention, the myeloid cells include at least one selected from monocytes, macrophages, and polymorphonuclear granulocytes.

[0042] Monocytes are the main myeloid cells in the bloodstream. Under the action of external stimuli, they can differentiate into antigen-presenting cells and activate subsequent immune responses. The inventors mainly use monocytes as the basis for cell vaccines. It should be noted that other types of myeloid cells, such as macrophages and polymorphonuclear granulocytes, can also be used as the basis for cell vaccines.

[0043] The fourth aspect of this invention provides the use of the modified gel described in the first aspect, the engineered myeloid cells described in the second aspect, and the engineered myeloid cells prepared by the preparation method described in the third aspect in the preparation of cell vaccines.

[0044] According to an embodiment of the present invention, the cell vaccine is an anti-tumor cell vaccine.

[0045] According to an embodiment of the present invention, the tumor includes at least one selected from melanoma, colon cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, and kidney cancer.

[0046] A fifth aspect of the present invention provides a cell vaccine. According to an embodiment of the present invention, the cell vaccine comprises engineered myeloid cells as described in the first aspect and / or engineered myeloid cells prepared by the preparation method described in the second aspect.

[0047] The sixth aspect of this invention provides the use of the modified gel described in the first aspect, the engineered myeloid cells described in the second aspect, and the engineered myeloid cells prepared by the preparation method described in the third aspect in the preparation of antitumor drugs.

[0048] According to an embodiment of the present invention, the tumor includes at least one selected from melanoma, colon cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, and kidney cancer.

[0049] A seventh aspect of the present invention provides a pharmaceutical composition. According to embodiments of the present invention, it comprises the modified gel described in the first aspect, or the engineered myeloid cells described in the second aspect, or the engineered myeloid cells prepared by the preparation method described in the third aspect.

[0050] According to an embodiment of the present invention, the pharmaceutical composition further comprises an immune checkpoint inhibitor.

[0051] According to an embodiment of the present invention, the immune checkpoint inhibitor includes at least one selected from anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIM-3, anti-TIGIT, and anti-CD47.

[0052] Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. Attached Figure Description

[0053] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0054] Figure 1 The preparation process of a surface-modified antigen gel (MAS) according to an embodiment of the present invention is shown;

[0055] Figure 2 The results of two-photon fluorescence imaging of the antigen and mouse bone marrow cells in Example 2 of this invention are shown.

[0056] Figure 3 The image shows the TMR-positive macrophages in each group after 6 days of treatment and injection in mice, as described in Example 3 of this invention. Changes in the number of dendritic cells (DCs) and monocytes, for each cell type, in Figure 3 The horizontal axis represents the groups from left to right: Blank gel group, S:GM+ group, and MAS:GM+ group. Blank gel refers to the control group without antigen and immunostimulant coating, S:GM+ group refers to the gel without antigen modification, and MAS:GM+ group refers to the gel injected into mice with the surface-modified antigen in Example 1.

[0057] Figure 4 This illustrates the changes in the number of TMR-positive dendritic cells and monocytes in the draining lymph nodes of mice treated with the methods described in Example 3 of this invention, 6 days after injection. For each cell type, [the following is a partial translation of the original text, which is not directly related to the preceding paragraph]. Figure 4The horizontal axis represents the groups from left to right: Blank gel group, S:GM+ group, and MAS:GM+ group.

[0058] Figure 5 This invention illustrates the effects of different immunostimulants contained in the gel on the number of monocytes and dendritic cells after mice were treated and injected for 6 days in Example 3 of the present invention.

[0059] Figure 6 The diagram shows the statistical results of the number of metastatic nodules in the lungs of healthy mice after different treatments and subsequent injection of melanoma cells B16-OVA in Example 4 of the present invention. In this diagram, Naive represents the control without vaccine injection; BolusVax represents the injection of a mixture of OVA peptide and adjuvant alone; SVax represents the injection of gel without modified antigen peptide; and MASVax represents the injection of gel with the modified antigen of the present invention.

[0060] Figures 7A-7C The figures show the tumor growth (7B) and mouse survival (7C) after subcutaneous injection of B16-OVA tumor cells into mice in Example 4 of the present invention, following different treatments. Figure 7A The vaccination process was demonstrated;

[0061] Figures 8A-8B The following figures illustrate the growth of tumors (8A) and the survival time of mice (8B) after subcutaneous injection of MC38 tumor cells into mice in Example 4 of the present invention, and the different treatments administered to the mice.

[0062] Figure 9 This invention illustrates the tumor growth and mouse survival after subcutaneous injection of MC38 tumor cells into mice and subsequent treatments. αPD-1 refers to the PD-1 monoclonal antibody.

[0063] Figure 10 The illustration shows the growth and survival of mice in each group after being re-inoculated with MC38 tumor cells in mice whose tumors had completely regressed, as described in Example 5 of this invention. Detailed Implementation

[0064] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0065] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0066] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0067] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined elsewhere in this document, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains.

[0068] In this document, the terms “comprising” or “including” are open-ended expressions, meaning that they include the contents specified in this invention, but do not exclude other aspects.

[0069] In this document, the terms “optionally,” “optionally,” or “optionally” generally refer to an event or condition that may, but may not, occur, and the description includes both cases in which the event or condition occurs and cases in which the event or condition does not occur.

[0070] In this document, the term "pharmaceutical composition" generally refers to a unit dosage form and can be prepared by any method well known in the pharmaceutical industry. All methods involve the step of combining the active ingredient with excipients that constitute one or more adjunct components. Typically, compositions are prepared by uniformly and sufficiently combining the active compound with liquid excipients, finely chopped solid excipients, or both.

[0071] In this document, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient in a suitable manner. The fusion protein or pharmaceutical composition of the present invention can be administered via any common route, as long as it can reach the intended tissue. Various routes of administration are contemplated, including intravenous injection, intramuscular injection, subcutaneous injection, intratumoral injection, etc., but the present invention is not limited to these exemplified routes of administration. Preferably, the compositions of the present invention are administered via subcutaneous injection or intratumoral injection.

[0072] In this document, the terms "cancer" or "tumor" can refer to any unregulated cell growth. Examples include, but are not limited to, non-small cell lung cancer, papillary thyroid carcinoma, glioblastoma multiforme, colon cancer, rectal cancer, lung cancer, head and neck cancer, kidney cancer, bladder cancer, breast cancer, ovarian cancer, liver cancer, bile duct cancer or sarcoma, acute myeloid leukemia, large cell neuroendocrine carcinoma, neuroblastoma, prostate cancer, neuroblastoma, pancreatic cancer, melanoma, head and neck squamous cell carcinoma, cervical cancer, skin cancer, glioma, esophageal cancer, oral squamous cell carcinoma, or gastric cancer, etc.

[0073] In this document, the term "treatment" means used to refer to achieving a desired pharmacological and / or physiological effect. This effect may be preventative in terms of complete or partial prevention of disease or its symptoms, and / or therapeutic in terms of partial or complete cure of disease and / or adverse effects caused by disease. As used herein, "treatment" covers diseases in mammals, particularly humans, including: (a) prevention of disease or the onset of disease in individuals susceptible to disease but not yet diagnosed with the disease; (b) inhibition of disease, such as blocking disease progression; or (c) relief of disease, such as reducing disease-related symptoms. As used herein, "treatment" encompasses any administration of a drug or compound to an individual to treat, cure, relieve, improve, reduce, or inhibit the individual's disease, including but not limited to administration of a drug containing a compound described herein to an individual in need.

[0074] In this article, the term "anti-tumor drugs" includes drugs that reduce the volume of solid tumors, inhibit the proliferation of tumor cells, alleviate clinical symptoms of tumors, and inhibit tumor recurrence.

[0075] According to a specific embodiment of the present invention, the present invention provides an antigen peptide-modified gel, comprising:

[0076] Methacrylate-modified hyaluronic acid;

[0077] Crosslinking agent;

[0078] Colony stimulating factors;

[0079] Immunostimulants

[0080] And antigenic peptides,

[0081] The gel contains a porous structure, the colony-stimulating factor and the immunostimulant are encapsulated inside the pores, and the antigenic peptide is loaded on the surface of the gel.

[0082] According to a specific embodiment of the present invention, the colony-stimulating factor is preferably GM-CSF, and the immunostimulant includes at least one selected from CpG, MDP, R848, cGAMP, Pam3CSK4, and PolyI:C, such as CpG, MDP, R848, cGAMP, etc. The immunostimulant in the present invention includes, but is not limited to, the above-mentioned types.

[0083] According to embodiments of the present invention, the crosslinking agent includes ammonium persulfate and N,N,N',N'-tetramethyldiethylamine. The present invention employs specific types of crosslinking agents, which, compared to other commonly used crosslinking agents, can further improve the stability of the antigen-gel backbone connection.

[0084] According to embodiments of the present invention, the immunostimulant includes at least one selected from STING agonists, NLR agonists, and TLR agonists. According to embodiments of the present invention, the NLR agonist includes, but is not limited to, NOD2, and other types of NLR agonists are also covered within the scope of protection of the present invention. According to embodiments of the present invention, the TLR agonist includes at least one selected from TLR1 / 2, TLR3, TLR4, TLR7 / 8, and TLR9, and the TLR agonists in the present invention include, but are not limited to, the types listed above.

[0085] According to an embodiment of the present invention, the gel is obtained by freeze polymerization of a mixture of methacrylate-modified hyaluronic acid, a crosslinking agent, a colony-stimulating factor, and an immunostimulant, and the antigenic peptide is loaded onto the surface of the gel by the following method:

[0086] (1) The gel obtained by freeze polymerization is reacted with compound A to obtain the first intermediate product;

[0087] (2) Obtain the thiol-modified antigenic peptide, mix the thiol-modified antigenic peptide with a thioctic acid monomer derivative, and perform a polymerization reaction to obtain a second intermediate product;

[0088] (3) Mix the first intermediate product and the second intermediate product to obtain a gel modified with the antigen peptide;

[0089] The structural formula of compound A is as follows:

[0090]

[0091] There are no particular restrictions on the conditions for freeze polymerization. For example, freeze polymerization can be carried out at -15℃ to 30℃, preferably at -20℃. There are no particular restrictions on the physical state of the gel itself, as long as it can stably encapsulate colony-stimulating factors and immunostimulants and provide a backbone for antigen loading.

[0092] According to an embodiment of the present invention, the lipoic acid monomer derivative is a compound of formula B, and the structural formula of compound B is:

[0093]

[0094] B

[0095] Wherein, R is selected from H, -COO - Alternatively, it may contain a guanidine substituent. In the structural formula of the lipoic acid monomer derivative provided by the present invention, R can be an uncharged group, a positively charged group, or a negatively charged group, and preferably a positively charged group. For example, R is preferably a guanidine substituent. This is because the lipoic acid monomer derivative containing a guanidine group is linked to the antigen, and the cell membrane surface is negatively charged. Through charge interaction, this is more conducive to the antigen entering the cell. According to an embodiment of the present invention, the lipoic acid monomer derivative is selected from at least one of the following:

[0096]

[0097] When myeloid cells migrate into the gel, their surface thiol groups can bind antigenic peptides, allowing these peptides to be loaded onto the cells. Simultaneously, the immunostimulants in the gel effectively activate the migrated cells, achieving in-situ engineering. Subsequent cell and animal experiments validated the effectiveness of this system and demonstrated the crucial role of monocytes in the immune response induced by this vaccine platform. The MAS platform simplifies the traditional cell vaccine manufacturing process and exhibits a significant anti-tumor immune response, showing promising application prospects. This type of myeloid cell-adoptive scaffold (MAS) effectively engineers myeloid cells and optimizes cell vaccine design strategies.

[0098] According to a specific embodiment of the present invention, the present invention provides a method for preparing engineered myeloid cells, the method comprising:

[0099] Myeloid cells are mixed with the modified gel described above. The thiol groups on the cell membrane surface of the myeloid cells undergo a thiol-disulfide bond exchange reaction with the modified gel, so that the antigenic peptides loaded on the surface of the modified gel are attached to the surface of the myeloid cells or enter the myeloid cells, in order to obtain engineered myeloid cells.

[0100] Engineered myeloid cells can be engineered in vitro or in situ in vivo. The gel, which contains antigens modified with various immunostimulants, attracts the migration of myeloid cells (such as monocytes) and causes them to differentiate into antigen-presenting cells. The activated monocytes loaded with antigens then migrate out of the gel and travel to the lymph nodes, which can efficiently induce anti-tumor immune responses from lymphocytes such as T cells.

[0101] The myeloid cells mentioned in this invention are not limited to monocytes, macrophages, or polymorphonuclear granulocytes, but may also be other types of myeloid cells.

[0102] According to a specific embodiment of the present invention, the present invention provides a cell vaccine comprising the engineered myeloid cells described above and / or the engineered myeloid cells prepared by the preparation method described in the second aspect.

[0103] Engineered myeloid cells with antigens modified on their surface can act as cellular vaccines to induce anti-tumor immune responses in lymphocytes such as T cells.

[0104] The antigens mentioned in this invention generally refer to antigenic peptides. There are no particular limitations on the type and size of antigenic peptides. All types of antigenic peptides known in the art that can be used as tumor antigens can be used in accordance with the method of this invention to achieve the engineered modification of myeloid cells and further obtain cell vaccines for the prevention of tumors.

[0105] According to a specific embodiment of the present invention, the present invention provides a pharmaceutical composition comprising the modified gel described above, or the engineered myeloid cells described above, or the engineered myeloid cells prepared by the preparation method described above.

[0106] According to embodiments of the present invention, the pharmaceutical composition further comprises an immune checkpoint inhibitor. There are no particular limitations on the type of immune checkpoint inhibitor; for example, it can be some commonly used immune checkpoint inhibitors in the art, such as anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-LAG-3, anti-TIM-3, anti-TIGIT, and anti-CD47. The pharmaceutical composition provided by the present invention can be used as an anti-tumor drug to treat tumors or alleviate tumor symptoms. There are no particular limitations on the types of tumors; for example, it can be solid tumors such as melanoma, colon cancer, breast cancer, pancreatic cancer, lung cancer, gastric cancer, and kidney cancer. The following will explain the scheme of this disclosure in conjunction with embodiments. Those skilled in the art will understand that the following embodiments are only for illustrating this disclosure and should not be considered as limiting the scope of this disclosure. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0107] Example 1: Preparation method of MAS

[0108] Figure 1 The preparation process of the surface-modified antigen gel (MAS) is shown below:

[0109] 1. Gel preparation

[0110] Methacrylate-modified hyaluronic acid (an ester compound formed by methacrylic acid and the hydroxyl groups in hyaluronic acid) was mixed with various active components (GM-CSF: 1.25 μg, immunostimulants: 50 μg each, R848 and cGAMP encapsulated in the gel in the experiment, antigen: 100 μg) and crosslinking agents (ammonium persulfate and N,N,N',N'-tetramethyldiethylamine). The different component ratios were: hyaluronic acid (2%, w / v), N,N,N',N'-tetramethyldiethylamine (0.5%, w / v), and ammonium persulfate (0.125%, w / v). The mixture was freeze-polymerized at -20°C to form a gel backbone.

[0111] 2. Gel modification

[0112] The resulting gel was further reacted with compound A, causing the carboxyl group in the hyaluronic acid to be converted into iodoacetamino group, yielding intermediate product 1. Then, the thiol-modified antigenic peptide was mixed with a lipoic acid monomer derivative, and the reaction was carried out under the following conditions to obtain a suitable degree of polymerization: the solution pH was adjusted to 7.5-8.5, and the reaction was carried out at -30℃ for 2.5 h, yielding intermediate product 2.

[0113] Intermediate product 1 was added to intermediate product 2 to terminate the polymerization, and finally a gel with surface-modified antigen was obtained.

[0114]

[0115] Example 2: Cellular uptake of antigenic peptides from gel surface

[0116] To investigate the uptake of gel surface antigens by myeloid cells, the surface-modified antigen gel obtained in Example 1 was modified with a fluorescent group, tetramethylrhodamine (TMR), for localization. The gel was then co-incubated with extracted mouse (C57BL / 6 female) bone marrow cells for 30 min, followed by two-photon fluorescence imaging. Figure 2 The cells were pre-labeled with CFSE, indicated by thin white arrows in the figure; the TMR-labeled antigenic peptides were indicated by white curved arrows; and the cells and antigenic peptides co-localized within the white dashed box were indicated by thick white arrows.

[0117] Example 3: Immunological evaluation of MAS

[0118] Following subcutaneous injection of MAS into female C57BL / 6 mice, immune cells were analyzed within the gel and in draining lymph nodes. The antigenic peptides in MAS were also first labeled with TMR, with an unmodified gel serving as a control (S). Six days after injection, a significant increase in TMR-positive macrophages, dendritic cells, and monocytes was observed in the MAS gel. Figure 3 This indicates that MAS can effectively promote phagocytosis of antigens by cells; at the same time, the number of TMR-positive cells in the draining lymph nodes was also significantly increased. Figure 4 This indicates that immune cells migrating from the gel can accumulate in the draining lymph nodes, initiating subsequent immune responses. Furthermore, the effects of loading the gel with different immunostimulants were investigated, and it was found that various adjuvants, including CpG, MDP, and R848, all had certain effects. Figure 5 ).

[0119] Example 4: Evaluation of the efficacy of MAS dual-cell vaccine

[0120] In the evaluation process, OVA peptides were used as antigenic peptides loaded in gels. First, the preventative effect of MAS was evaluated: healthy mice were subcutaneously injected with MAS (days 0 and 14), and on day 60, melanoma cells B16-OVA were intravenously injected. The experimental group mice showed almost no traces of metastatic tumors in their lungs, indicating that this MAS system can effectively prevent tumor development. Figure 6 ).

[0121] The anti-tumor effect was then evaluated using a tumor-bearing model: after subcutaneous injection of B16-OVA tumors into mice, the mice were administered MAS vaccine twice or multiple doses of a single vaccine (BolusVax, a direct mixture of OVA peptide and adjuvant). The results showed that, compared with other groups, MAS effectively inhibited tumor growth and prolonged the survival of mice. Figures 7A-7C Further, the antitumor properties of MAS were verified using the MC38 tumor model: the antigen in MAS was replaced with the new antigen adpgk peptide of MC38 colon cancer cells, and the results also verified that MAS can effectively induce an antitumor immune response. Figures 8A-8B ).

[0122] Example 5: Evaluation of the antitumor effect of MAS in combination with immune checkpoint inhibitors

[0123] The efficacy of MAS combined with an immune checkpoint inhibitor was further evaluated in an MC38 tumor model. MAS was injected 8 days after MC38 tumor inoculation, followed by two injections every 7 days; PD-1 monoclonal antibody treatment was administered on days 3 and 6 after each immunization. Results showed that the combination therapy significantly inhibited tumor growth, and 80% of the tumors in the experimental group completely regressed. Figure 9 Mice whose tumors had completely regressed were re-inoculated with tumor cells, and none of the mice showed tumor recurrence. Figure 10 This further validates that combination therapy can generate long-term immune surveillance, preventing tumor recurrence and metastasis.

[0124] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0125] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A modified gel, characterized in that, Comprise: Methacrylate-modified hyaluronic acid; Crosslinking agent; Colony stimulating factor; Immune stimulant, And antigen peptide, Wherein, the gel contains pore structure, the colony stimulating factor and the immune stimulant are wrapped inside the pore structure, and the antigen peptide is loaded on the surface of the gel, The gel is obtained by freezing polymerization after mixing the methacrylate-modified hyaluronic acid, the crosslinking agent, the colony stimulating factor and the immune stimulant; The antigen peptide is loaded on the surface of the gel by the following method: (1) The gel obtained by freezing polymerization is reacted with a compound of formula A to obtain a first intermediate product; (2) Obtain a thiol-modified antigen peptide, mix the thiol-modified antigen peptide with a lipoic acid monomer derivative, and carry out a polymerization reaction to obtain a second intermediate product; (3) Mix the first intermediate product and the second intermediate product to obtain an antigen peptide-modified gel; Wherein, the compound of formula A has the following structure: A， Wherein, the colony stimulating factor is GM-CSF, the immune stimulant is R848 and cGAMP, the crosslinking agent is ammonium persulfate and N, N, N', N'-tetramethyl diethylamine, and the antigen peptide is OVA polypeptide with the amino acid sequence SIINFEKL, The structure of the lipoic acid monomer derivative is: 。 2. Use of the modified gel of claim 1 in the preparation of a vaccine, wherein the vaccine is an anti-tumor vaccine; The tumor is melanoma or colon cancer.

3. Use of the modified gel of claim 1 in the preparation of an anti-tumor drug, The tumor is melanoma or colon cancer.

4. A pharmaceutical composition, characterized by, Comprise the modified gel of claim 1.

5. The pharmaceutical composition of claim 4, wherein, The pharmaceutical composition further comprises an immune checkpoint inhibitor; The immune checkpoint inhibitor is anti-PD-1.

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