A tumor in-situ vaccine based on an immune-activated high molecular material and preparation and application thereof

Abstract

The application belongs to the technical field of tumor vaccines, and discloses a tumor in-situ vaccine based on an immunologically active high molecular material and a preparation and application thereof. The tumor in-situ vaccine has a structure of formula I, wherein the -NH- in the structure is an amine group -NH2 from the end of the PAMAM polymer, n is 45, the PAMAM polymer is a fourth-generation PAMAM polymer, and m is an integer of 5-10. The application further discloses a preparation method of the tumor in-situ vaccine. The tumor in-situ vaccine based on the immunologically active high molecular material is used as an anti-tumor tumor in-situ vaccine. The tumor in-situ vaccine has the abilities of inducing immunogenic cell death, activating BMDCs and repolarizing macrophages, and can improve the tumor immune microenvironment. Meanwhile, the tumor in-situ vaccine has a strong anti-tumor effect, can produce a strong antigen-specific cellular immune effect to kill tumors, and can form an immune memory.

Description

Technical Field

[0001] This invention belongs to the technical field of medical materials, specifically relating to an in situ tumor vaccine constructed based on immunologically active polymer materials and its preparation and application. Background Technology

[0002] Therapeutic tumor vaccines induce an antigen-specific immune response in the body through tumor antigens. Their purpose is to induce tumor regression or eliminate residual microtumors after surgery, establish long-term anti-tumor immune memory, and prevent tumor metastasis and recurrence. Tumor vaccines mainly consist of two important parts: antigens and adjuvants. Antigens are often composed of protein molecules overexpressed or specifically expressed on tumor cells, inactivated tumor cells, or cellular components; their form can be nucleic acids, peptides / proteins, whole tumor cells, or tumor cell lysates. Adjuvants are non-specific immunomodulatory substances used to enhance the antigen-induced anti-tumor immune response. The composition and function of antigens and adjuvants are key considerations in designing highly effective tumor vaccines.

[0003] In inducing a specific anti-tumor immune response, each step can potentially affect the efficacy of a tumor vaccine. Firstly, the immunogenicity of the tumor antigen determines the effectiveness of the anti-tumor immune response. However, most solid tumors themselves have low immunogenicity, and tumor vaccines based on exogenous tumor antigens such as nucleic acids, peptides / proteins often fail to elicit a strong immune response due to insufficient antigen immunogenicity. Secondly, the types of adjuvants currently available are limited, and their immunoactivation effects are restricted by factors such as solubility and pharmacokinetics. Furthermore, the tumor microenvironment is rich in immunosuppressive cells and factors, making it difficult for T cells to infiltrate and kill tumor cells.

[0004] Compared with other tumor vaccines, in situ tumor vaccines have many advantages: 1) Simple preparation process, no need to screen antigens and target audiences. 2) Overcoming tumor heterogeneity, possessing both personalization and universality. 3) Overcoming dynamic immune tolerance. Immunogenic cell death (ICD) is an effective way to establish in situ tumor vaccines to provide endogenous personalized antigens, which can enhance the immunogenicity of tumors. When tumor cells undergo ICD, they express and release a series of damage-associated molecular patterns (DAMPs), including the extravasation of calreticulin (CALR) from the endoplasmic reticulum to the cell membrane surface, secretion of adenosine triphosphate (ATP), and release of high-mobility group box 1 (HMGB1). These DAMPs can recruit and activate dendritic cells (DCs), promoting the uptake and processing of tumor antigens by DCs, thereby inducing antigen-specific T cells to kill tumor cells. Currently, common strategies for inducing ICD include radiotherapy, photodynamic therapy (PDT), and some chemotherapy drugs such as doxorubicin and mitoxantrone.

[0005] Toll-like receptor (TLR) agonists can initiate innate immunity and help activate adaptive immunity. Therefore, TLR agonists have attracted considerable attention in the field of tumor vaccine adjuvants. Resiquimod (R848), as a TLR7 / 8 agonist, can not only activate antigen-presenting cells but also effectively repolarize macrophages from the tumor-promoting M2 type to the tumor-suppressive M1 type, improving the immunosuppressive microenvironment within the tumor, thus showing great potential for application in anti-tumor immunotherapy. However, as a small molecule drug, R848 has poor water solubility and is easily metabolized and cleared, resulting in low bioavailability. Furthermore, systemic exposure to the drug can cause significant toxicity, thus limiting its application in tumor immunotherapy.

[0006] This invention utilizes D47, a polymeric material with ICD-inducing effects, as a carrier to bond adjuvant molecule R848, constructing a therapeutic in situ tumor vaccine, D47-R848. Specifically, the ICD material D47 can induce tumor cell apoptosis and provide personalized tumor antigens in situ, while R848, while serving as an effective adjuvant for the vaccine, can repolarize tumor immune microenvironments (TAMs) in the tumor microenvironment, activating and reversing the inhibitory tumor immune microenvironment, thereby enhancing the efficacy of the tumor vaccine. Summary of the Invention

[0007] The purpose of this invention is to provide an in situ tumor vaccine based on an immunomodulatory polymer and its preparation method. This invention utilizes the reaction of D47 surface amino groups with HSEA-R848. The in situ tumor vaccine of this invention not only has the ability to induce immunogenic cell death, but also has the function of activating BMDCs and repolarizing macrophages, thereby improving the tumor immune microenvironment; simultaneously, it exhibits a strong anti-tumor effect, generating a powerful antigen-specific cellular immune response to kill tumor cells and forming immune memory.

[0008] Another object of the present invention is to provide the application of the above-mentioned tumor in situ vaccine. The polymer of the present invention has the effect of inducing immunogenic cell death and exhibiting a strong anti-tumor activity. The tumor in situ vaccine of the present invention is used as an anti-tumor tumor vaccine for the preparation of anti-tumor products.

[0009] The objective of this invention is achieved through the following technical solution:

[0010] A tumor in situ vaccine constructed based on an immunologically active polymer material, the structure of which is as follows:

[0011]

[0012] The -NH- group connected to PAMAM in the structure comes from the amino group -NH2 at the end of the PAMAM polymer, and n is 45.

[0013] PAMAM polymer is a fourth-generation PAMAM polymer.

[0014] m is an integer between 5 and 10.

[0015] The method for preparing the tumor in situ vaccine based on the immunologically active polymer material includes the following steps: reacting the immunologically active polymer material with HSEA-R848 to obtain the tumor in situ vaccine;

[0016] The structure of the immunomodulatory polymer is PAMAM-(NH-C(O)-O-CH2CH2-R). n R is -NH- comes from the amino group -NH2 at the end of the PAMAM polymer; n is 45;

[0017] PAMAM polymer is a fourth-generation PAMAM polymer.

[0018] The specific structure of the immunomodulatory polymer (i.e., D47) is as follows:

[0019]

[0020] The structure of HSEA-R848 is as follows

[0021]

[0022] The reaction is carried out in an organic solvent as the reaction medium, and the organic solvent is one or more of dimethyl sulfoxide or N,N-dimethylformamide.

[0023] The reaction is carried out in the presence of a catalyst, which is one or more of diisopropylethylamine, triethylamine, and pyridine.

[0024] The molar ratio of the immunomodulatory polymer (i.e., D47) to HSEA-R848 is 1:10-30;

[0025] The reaction temperature is 40–60°C, and the reaction time is 24–72 h.

[0026] The tumor in situ vaccine based on immunomodulatory polymer materials is prepared by reacting the amino groups on the surface of D47 with HSEA-R848, and the reaction equation is as follows:

[0027]

[0028] The tumor in situ vaccine constructed based on immunologically active polymer materials is used as an anti-tumor tumor in situ vaccine.

[0029] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0030] The present invention, D47-R848, binds D47, which can provide in situ personalized antigens, to an R848 derivative with an adjuvant effect, thus maintaining not only the original function of D47 but also the biological activity of R848. D47-R848 not only has the ability to induce immunogenic cell death but also the function of activating BMDCs and repolarizing macrophages, thereby improving the tumor immune microenvironment.

[0031] The tumor in situ vaccine constructed from immunologically active polymer materials of this invention, as a personalized in situ vaccine, has shown strong anti-tumor effects in various types of tumor models, generating a powerful antigen-specific cellular immune response to kill tumor cells and form immune memory. Therefore, it has enormous application potential in fields such as biomedicine. Attached Figure Description

[0032] Figure 1 The hydrogen NMR spectrum of D47-R848;

[0033] Figure 2 Electron micrograph of D47-R848;

[0034] Figure 3 To validate at the cellular level that D47-R848 induces calreticulin eversion in 4T1 cells;

[0035] Figure 4 To validate at the cellular level that D47-R848 induces HMGB1 release in 4T1 cells;

[0036] Figure 5 To verify at the cellular level that D47-R848 induces ATP secretion in 4T1 cells;

[0037] Figure 6 Figure 1 shows the results of D47-R848 activation of BMDCs at the cellular level; (a) CD80 levels in different experimental groups were detected by flow cytometry. + CD86 + (b) Changes in the proportion of BMDCs; (c) Detection of MHC-II expression in different experimental groups by flow cytometry.

[0038] Figure 7 Figure 1 shows the results of validating D47-R848 repolarized BMDMs at the cellular level; (a) CD86 expression in different experimental groups was detected by flow cytometry. + Cells in CD11b + (b) Changes in cell proportions; (c) Detection of CD206 expression in different experimental groups by flow cytometry + Cells in CD11b + Changes in cell proportions;

[0039] Figure 8 To validate the therapeutic effect of D47-R848 in a 4T1 orthotopic breast cancer model at the animal level; (a) Schematic diagram of the vaccine injection process; (b) Tumor growth inhibition in different treatment groups; (c) Changes in body weight of mice in different treatment groups;

[0040] Figure 9 To validate the therapeutic effect of D47-R848 in the MC38 subcutaneous colorectal cancer model at the animal level; (a) Schematic diagram of the vaccine injection process; (b) Tumor growth inhibition in different treatment groups; (c) Statistical graph of tumor quality in mice in each group after treatment;

[0041] Figure 10 To validate the enhancement of antigen-specific T cell response by D47-R848 in animals; (a, b) ELISPOT analysis of IFN-γ spots formed in spleen lymphocytes after in vitro 4T1 tumor fluid restimulation; where (a) is a bar chart and (b) is a visual chart. Detailed Implementation

[0042] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto.

[0043] Example 1: Synthesis of D47-R848, an in situ tumor vaccine constructed based on immunomodulatory polymer materials.

[0044] D47-R848 is prepared by reacting the amino groups on the surface of D47 with HSEA-R848.

[0045] Preparation of D47: See the dendritic macromolecule G4-PAMAM-C7A in the reference patent (application number 201810820872.0, title: A modified dendritic PAMAM polymer and its preparation method and application). 45 Preparation of .

[0046] Structure of HSEA-R848:

[0047] Preparation of D47-R848:

[0048] (1) Add 25 mg of D47 and 18 mg of HSEA-R848 (molar ratio of the two substances is 1:15) to 1.5 ml of anhydrous DMSO and transfer it to a reaction vessel. Add 20 μL of DIPEA (N,N-diisopropylethylamine) to the reaction vessel and react in an oil bath at 50 °C for 48 hours.

[0049] (2) After the reaction was completed, the product was concentrated to 0.5 mL under vacuum. The product was separated and collected by Sephadex LH20 gel column with methanol as the mobile phase. The methanol was removed under reduced pressure to obtain D47-R848.

[0050] MRI results of D47-R848 ( 1 (H NMR spectrum) as shown Figure 1 As shown. Electron micrograph of D47-R848 is shown below. Figure 2 As shown.

[0051] Example 2: Cellular-level verification of D47-R848-induced calreticulin eversion in 4T1 cells

[0052] The ability of D47-R848 to induce cadherin extraversion in 4T1 cells was detected by flow cytometry (FACs). 4T1 tumor cells were seeded in 24-well plates at a cell density of 1 × 10⁻⁶ cells / well. 5 Cells / well. D47-R848 cells were co-incubated with 4T1 tumor cells for 4 h. Cells were labeled with CALR fluorescent antibody and stained with PI. Flow cytometry was used to detect and count the expression of CALR on the surface of live cells. Experimental results are as follows: Figure 3 As shown, D47-R848 can effectively induce calreticulin eversion in 4T1 cells.

[0053] Example 3: Cellular-level verification of D47-R848-induced release of HMGB1 from 4T1 cells

[0054] The ability of D47-R848 to induce the release of HMGB1 from 4T1 cells was detected by ELISA. 4T1 cells were seeded in 24-well plates at a cell density of 1 × 10⁻⁶ cells / well. 5 Cells / well. D47-R848 cells were co-incubated with 4T1 tumor cells for 24 h. Cell supernatant was collected, and cell debris and impurities were removed by centrifugation. The concentration of HMGB1 in the supernatant was detected using an ELISA kit. Experimental results are as follows: Figure 4 As shown, D47-R848 can effectively induce 4T1 cells to release HMGB1.

[0055] Example 4: Cellular-level verification of D47-R848 inducing 4T1 cells to secrete 5'-adenosine triphosphate (ATP)

[0056] The ability of D47-R848 to induce ATP secretion in 4T1 cells was detected using an ATP assay kit. 4T1 cells were seeded in 24-well plates at a cell density of 1 × 10⁶ cells / well. 5 Cells / well. D47-R848 cells were co-incubated with 4T1 tumor cells for 24 h. Cell supernatant was collected and centrifuged to remove cell debris and impurities. 50 μL of ATP detection working solution was added to an opaque 96-well plate, allowed to stand for 3-5 min to deplete background, and then 50 μL of sample was added. Detection was performed using a multi-functional microplate reader. Experimental results are as follows: Figure 5 As shown, D47-R848 can effectively induce 4T1 cells to secrete ATP.

[0057] Example 5: Cellular-level verification of D47-R848 activation of BMDCs

[0058] Flow cytometry (FACs) was used to verify the ability of D47-R848 to induce BMDC activation. We used mouse bone marrow-derived dendritic cells (BMDCs) for experimental verification. Extracted bone marrow cells were seeded in 24-well plates at a cell density of 5 × 10⁶ cells / well. 5 / well. BMDCs were induced using cytokines 20 ng / mL mouse GM-CSF and 5 ng / mL mouse IL-4. BMDCs cultured to day 6 were stimulated with D47-R848 for 24 h, and cells were labeled with specific fluorescent antibodies. CD80 and CD86 expression was detected using FACs. Experimental results are shown below. Figure 6 As shown, both the D47-R848 and R848 treatment groups can enhance the expression of BMDC activation markers such as CD86, CD80 and major histocompatibility complex class II (MHC-II), demonstrating that R848 can still effectively activate BMDCs in vitro after modification.

[0059] Figure 6Figure 1 shows the results of D47-R848 activation of BMDCs at the cellular level; (a) CD80 levels in different experimental groups were detected by flow cytometry. + CD86 + (a) Changes in the proportion of BMDCs; (b) Detection of the expression of antigen-presenting molecule MHC-II in different experimental groups by flow cytometry.

[0060] Example 6: Cellular-level validation of D47-R848 repolarized BMDMs

[0061] Flow cytometry (FACs) was used to verify the repolarization ability of D47-R848-induced BMDMs. Mouse bone marrow-derived macrophages (BMDMs) were used for experimental verification. BMDMs were induced and cultured using 10 ng / mL mouse M-CSF. BMDMs cultured for 6 days were seeded into 24-well plates at a cell density of 1 × 10⁶ cells / well. 5 Cells were treated with 20 ng / mL IL-4 for 24 h to induce BMDMs to polarize to M2. Then, they were treated with D47-R848 for 24 h to induce M2 repolarization to M1. Simultaneously, stimulation with 20 ng / mL IFN-γ and 100 ng / mL LPS for 24 h served as a positive control for M1. After repolarization, cells were collected, labeled with specific fluorescent antibodies, and CD86 and CD206 expression was detected using FACs. Experimental results are as follows: Figure 7 As shown, compared with the M2 group, D47-R848 significantly increased the proportion of macrophages expressing CD86 and significantly decreased the proportion of macrophages expressing CD206. This indicates that D47-R848 can effectively polarize the pro-tumor M2 macrophages into anti-tumor M1 macrophages. Furthermore, the repolarization effect of D47-R848 is comparable to that of free R848. In summary, these results demonstrate that modified R848 bound to D47 retains its ability to repolarize macrophages in vitro.

[0062] Figure 7 Figure 1 shows the results of validating D47-R848 repolarized BMDMs at the cellular level; (a) CD86 expression in different experimental groups was detected by flow cytometry. + Cells in CD11b + (b) Changes in cell proportions; (c) Detection of CD206 expression in different experimental groups by flow cytometry + Cells in CD11b + Changes in cell proportions.

[0063] Example 7: Animal-level verification of the antitumor effect of D47-R848

[0064] We validated the therapeutic antitumor effect of D47-R848 as a universally applicable personalized in situ tumor vaccine in two mouse tumor models.

[0065] Model 1: 4T1 in situ breast cancer model

[0066] This experiment used 6-8 week old female BALB / c mice, injecting 500,000 4T1 tumor cells into the mammary pads to construct a 4T1 orthotopic breast cancer model. The experimental groups were: PBS group, R848 group, D47 group, and D47-R848 group. The vaccination flowchart is shown below. Figure 8 As shown in (a). The injection dose of R848 was 20 μg / animal, and the injection dose of D47 was 200 μg / animal. The injection dose of D47-R848 was the same as that of R848. The drugs were injected via tail vein once every three days for a total of three times. Tumor growth was observed after treatment. Experimental results are shown below. Figure 8 As shown in (b,c), compared to other groups, D47-R848, as an in situ tumor vaccine, can effectively inhibit tumor growth.

[0067] Model 2: MC38 subcutaneous colorectal cancer model

[0068] This experiment used 6-8 week old female C57BL / 6 mice. One million MC38 tumor cells were subcutaneously injected into the right back of the mice to establish an MC38 subcutaneous colorectal cancer model. The experimental groups were: PBS group, R848 group, D47 group, and D47-R848 group. The vaccination flowchart is shown below. Figure 9 As shown in (a). The injection dose of R848 was 10 μg / animal, and the injection dose of D47 was 100 μg / animal. The injection dose of D47-R848 was the same as that of R848. The drugs were injected via tail vein every three days for a total of three times. Tumor growth was observed after treatment. The experimental results are as follows: Figure 9 As shown in (b,c), compared with other groups, D47-R848, as an in situ tumor vaccine, can effectively inhibit tumor growth.

[0069] In summary, the binding of R848 effectively improved the therapeutic effect of D47 and achieved immune enhancement.

[0070] Figure 8 To validate the therapeutic effect of D47-R848 in a 4T1 orthotopic breast cancer model at the animal level; (a) Schematic diagram of the vaccine injection process; (b) Tumor growth inhibition in different treatment groups; (c) Changes in body weight of mice in different treatment groups;

[0071] Figure 9To validate the therapeutic effect of D47-R848 in the MC38 subcutaneous colorectal cancer model at the animal level; (a) Schematic diagram of the vaccine injection process; (b) Tumor growth inhibition in different treatment groups; (c) Statistical graph of tumor quality in mice in each group after treatment.

[0072] Example 8: Animal-level verification of D47-R848 enhancing antigen-specific T cell responses

[0073] Enzyme-linked immunosorbent assay (ELISPOT) was used to verify the enhancement of cellular immune response in mice after treatment with D47-R848 as an in situ vaccine. First, tumor cell lysates were prepared. 4T1 tumor cells in logarithmic growth phase were expanded and cultured in vitro. Cells were collected, centrifuged, and washed with sterile 1×PBS buffer (pH 7.4). The cells were resuspended in sterile water and lysed on ice for 30 min. The cells were subjected to six freeze-thaw cycles in liquid nitrogen and a 37°C water bath, followed by centrifugation at 12000 rpm for 5 min. The supernatant was collected, and protein content was quantitatively determined using a BCA assay kit. In the 4T1 in situ breast cancer treatment experiment, on day 7 after the last tail vein injection, mouse spleens were harvested, and splenic immune cells were isolated. 100 μL of cell culture medium and 1 million splenic immune cells were added to each well of an ELISPOT antibody-coated plate, stimulated with 100 μg / mL of 4T1 tumor cell lysate, and incubated at 37°C for 48 h. After 48 hours, the plate was read after antibody staining and development. The experimental results are as follows: Figure 10 As shown, compared with other groups, the number of T cells secreting IFN-γ was significantly increased in the D47-R848 group.

[0074] Figure 10 To validate the enhancement of antigen-specific T cell response by D47-R848 in animals; (a, b) ELISPOT analysis of IFN-γ spots formed in spleen lymphocytes after in vitro 4T1 tumor fluid restimulation; where (a) is a bar chart and (b) is a visual chart.

Claims

1. The application of an in situ tumor vaccine constructed based on an immunologically active polymer material in the preparation of antitumor products, characterized in that: The structure of the tumor in situ vaccine is shown in Formula I: The -NH- group attached to PAMAM in the structure comes from the amino group -NH2 at the end of the PAMAM polymer, with n being 45; PAMAM polymer is a fourth-generation PAMAM polymer; m is 7; The anti-tumor treatment mentioned is for breast cancer or colorectal cancer.

2. The application according to claim 1, characterized in that, The preparation of the tumor in situ vaccine includes the following steps: reacting an immunologically active polymeric material with HSEA-R848 to obtain the tumor in situ vaccine; The structure of the immunomodulatory polymer is PAMAM-(NH-C(O)-O-CH2CH2-R). n , R is ; -NH- comes from the amino group at the end of the PAMAM polymer -NH2; n is 45; PAMAM polymer is a fourth-generation PAMAM polymer; The structure of HSEA-R848 is as follows: 。 3. The application according to claim 2, characterized in that: The molar ratio of the immunomodulatory polymer to HSEA-R848 is 1:10-30; The reaction temperature is 40~60℃, and the reaction time is 24~72 h.

4. The application according to claim 2, characterized in that, The reaction is carried out in the presence of an organic solvent as the reaction medium and under the action of a catalyst.

5. The application according to claim 4, characterized in that: The organic solvent is one or more of dimethyl sulfoxide or N,N-dimethylformamide; The catalyst is one or more of diisopropylethylamine, triethylamine, and pyridine.

6. The application according to claim 1, characterized in that: The tumor in situ vaccine constructed based on immunologically active polymer materials is used as an in situ tumor vaccine against breast cancer or colorectal cancer.

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