A hydrogel vaccine system for combined removal of tumors and senescent cells, a hydrogel vaccine, and a preparation method and application thereof

Abstract

The present invention discloses a hydrogel vaccine system for combined removal of tumor and senescent cells, a hydrogel vaccine, and a preparation method and application, which belong to the technical field of tumor immunotherapy. The present invention provides a hydrogel vaccine system for enhanced tumor immunotherapy that combines removal of senescent cells and tumor cells, and the hydrogel system simultaneously loads: 1) vesicles derived from senescent tumor cell membranes, 2) vaccine adjuvants, and 3) DC cell chemokines and / or activation factors. The present invention constructs a combined vaccine that simultaneously removes senescent cells and tumor cells, relieves the inhibition of senescent cells on tumor immunity, and enhances the effect of tumor vaccines. The vaccine strategy has broad prospects for clinical application and is expected to revolutionize the direction of tumor immunotherapy, providing a new perspective for enhancing the effect of tumor immunotherapy.

Description

Technical Field

[0001] The present invention belongs to the technical field of immunotherapy, and specifically relates to a hydrogel vaccine system for combined removal of tumors and senescent cells, a hydrogel vaccine, and a preparation method and application thereof. Background Art

[0002] Cancer originates from malignant lesions of epithelial tissues. It is a complex process with multiple factors and steps. Its causes are closely related to heredity, infection, occupational exposure, environmental pollution, unreasonable lifestyle, etc. During the lesion process, the spread of cancer cells can damage cells outside the lesion site and eventually lead to the death of the patient.

[0003] Immunotherapy is an emerging method for tumor treatment. However, during tumor evolution, stress such as free radical damage causes senescent phenotypes in cells such as fibroblasts in the tumor. Senescent cells secrete a class of senescent phenotype-related molecules into immune cells in the tumor or draining lymph nodes, inhibiting immune function in multiple ways, thus delaying or ineffective the effect of immunotherapy.

[0004] Therefore, before or during tumor immunotherapy, cells with a senescent phenotype need to be eliminated, but there is currently no corresponding drug research. Summary of the invention

[0005] The purpose of the present invention is to provide a tumor immunotherapy hydrogel vaccine system, hydrogel vaccine, preparation method and application for combined removal of senescent and tumor cells, thereby enhancing tumor immunity effects while removing senescent cells.

[0006] The present invention provides a hydrogel vaccine system for tumor immunotherapy, wherein the hydrogel vaccine system is simultaneously loaded with DC cell chemotactic factors and / or activation factors, vaccine adjuvants, and vesicles derived from senescent tumor cell membranes.

[0007] Preferably, the DC cell chemokine and / or activation factor includes GM-CSF.

[0008] Preferably, the vaccine adjuvant comprises R848 or the STING agonist cGAMP.

[0009] Preferably, the tumor cell types induced to undergo senescence include solid tumor cells.

[0010] Preferably, the method for preparing induced senescent tumor cells comprises inducing tumor cell senescence by stress injury, squeezing and collecting cell membranes of the induced senescent tumor cells to prepare vesicles.

[0011] The present invention also provides a method for constructing the hydrogel vaccine system, comprising the following steps: initiating and dissolving GelMA, mixing it with Nanoclay, and incubating it at 50° C. in the dark for 2 hours to obtain GelMA / Nanoclay;

[0012] The hydrogel vaccine system is obtained by mixing GelMA / Nanoclay with DC cell chemotactic factors and / or activation factors and vesicles derived from senescent tumor cell membranes pre-loaded with vaccine adjuvants.

[0013] The present invention also provides a method for preparing a hydrogel vaccine for tumor immunotherapy, comprising irradiating the hydrogel vaccine system under ultraviolet light for 30 seconds to obtain the hydrogel vaccine.

[0014] Preferably, the wavelength of the ultraviolet light is 405nm and the power is 1W / cm 2 .

[0015] The present invention also provides a hydrogel vaccine prepared by the above preparation method.

[0016] The present invention also provides the use of the hydrogel vaccine system or the hydrogel vaccine in preparing tumor immunotherapy drugs.

[0017] Beneficial effects: The present invention provides a hydrogel vaccine system for tumor immunotherapy ( Figure 1 ), the hydrogel system is simultaneously loaded with: 1) vesicles derived from senescent tumor cell membranes pre-loaded with vaccine adjuvants, 2) DC cell chemokines and / or activation factors. The present invention uses hydrogels to release GM-CSF slowly to recruit and activate APCs, thereby avoiding the problem of difficult immune cell infiltration of tumor tissues; antigens of senescent tumor cells (with characteristics of tumor cells and senescent cells) loaded in the hydrogels can be phagocytosed and presented by APCs, providing antigens for the simultaneous removal of tumors and senescent cells; immune adjuvants further activate APCs, thereby activating T cell immunity against tumor cells and senescent cells, and the removal of senescent cells further relieves the suppression of immunity by tumors and enhances tumor immunity. The present invention constructs a combined vaccine for the removal of senescent cells and tumor cells, relieves the suppression of tumor immunity by senescent cells, and improves the effect of tumor vaccines. This vaccine strategy has broad prospects for clinical application and is expected to revolutionize the direction of tumor immunotherapy, providing a new perspective for improving the effect of tumor immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram for constructing hydrogel vaccines;

[0019] Figure 2 The efficiency of senescent cell clearance in tumor tissue after hydrogel vaccine treatment;

[0020] Figure 3CD8 tumor-infiltrating lymphocytes after hydrogel vaccine therapy + T cells and CD4 + T cell ratio;

[0021] Figure 4 The expression of pro-inflammatory cytokines Il-2, Tnf-α and Ifn-γ mRNA after hydrogel vaccine treatment;

[0022] Figure 5 Tumor volume of CT26.WT colon cancer mice in the control group and hydrogel vaccine treatment group;

[0023] Figure 6 These are the survival curves of CT26.WT colon cancer mice in the control group and the hydrogel vaccine treatment group. DETAILED DESCRIPTION

[0024] The present invention provides a hydrogel vaccine system for tumor immunotherapy, wherein the hydrogel vaccine system is simultaneously loaded with DC cell chemotactic factors and / or activation factors, and senescent tumor cell membrane-derived vesicles pre-loaded with vaccine adjuvants.

[0025] The hydrogel vaccine system of the present invention is preferably loaded based on GelMA / Nanoclay, and the preparation method of GelMA / Nanoclay preferably includes initiating and dissolving GelMA, mixing it with Nanoclay, and incubating it at 50°C in the dark for 2 hours to obtain GelMA / Nanoclay. The present invention first dissolves GelMA in 0.25% (w / v) initiator LAP (LithiumPhenyl-2,4,6-trimethylbenzoylphosphinat), and dissolves it in a light-proof water bath at 60°C to 70°C for 20 to 30 minutes. The added mass ratio of GelMA to Nanoclay of the present invention is preferably 0.1g:0.06g, and incubate at 50°C in the dark for 2 hours to obtain GelMA / Nanoclay.

[0026] The GelMA / Nanoclay of the present invention is simultaneously loaded with 1) vesicles derived from senescent tumor cell membranes pre-loaded with vaccine adjuvants, and 2) DC cell chemokines and / or activation factors. The types of tumor cells induced to age according to the present invention preferably include solid tumor cells, such as colon cancer cells as an example in the embodiment, but they cannot be identified as the entire protection scope of the present invention. The method for preparing senescent tumor cells according to the present invention preferably includes inducing tumor cell senescence by stress damage, squeezing the senescent tumor cells and collecting the cell membranes to make vesicles. The cell membrane fragments collected by the method of the present invention have both tumor cell antigens and senescent cell antigens. The stress damage of the present invention preferably includes oxidative stress, chemotherapy or beta rays, etc. The cells induced to age according to the present invention are obtained by trypsin digestion. After the cell suspension was centrifuged at 4°C and 500g for 5 min, the supernatant was removed and the cell pellet was suspended in a hypotonic buffer (hypotonic solution formula: 30mM Tris-HCL (pH7.5), 225mM D-mannitol, 75mM sucrose, 0.2mM EGTA and protease and phosphatase inhibitor cocktail). The cell suspension was subjected to 20 times of Dounce homogenizer to destroy the cells. The suspension was then centrifuged at 4°C and 2500g for 15 min, and the cell debris was discarded; after another centrifugation at 4°C and 12000g for 20 min, the cell debris was discarded. After filtering with a 0.22μm filter, the supernatant was transferred, ultracentrifuged at 4°C and 100000g for 4 hours, the supernatant was removed, and the pellet was resuspended in PBS to obtain the cell membrane vesicles.

[0027] The DC cell chemokine and / or activation factor of the present invention preferably includes GM-CSF; the vaccine adjuvant preferably includes R848 or STING agonist cGAMP. In the present invention, the mass ratio of GM-CSF to GelMA is 60-100ng:0.1g; the mass ratio of R848 to cell membrane is 10-30μg:300μg; the mass ratio of cell membrane to GelMA is 300-500μg:0.1g.

[0028] The present invention also provides a method for constructing the hydrogel vaccine system, comprising the following steps: initiating and dissolving GelMA, mixing it with Nanoclay, and incubating it at 50° C. in the dark for 2 hours to obtain GelMA / Nanoclay;

[0029] The hydrogel vaccine system is obtained by mixing GelMA / Nanoclay with DC cell chemotactic factors and / or activation factors and vesicles derived from senescent tumor cell membranes pre-loaded with vaccine adjuvants.

[0030] The preparation method of GelMA / Nanoclay of the present invention is preferably the same as that described above, and will not be described in detail herein. In the present invention, the above raw materials are preferably mixed and shaken to obtain the hydrogel vaccine system.

[0031] The present invention also provides a method for preparing a hydrogel vaccine for tumor immunotherapy, comprising irradiating the hydrogel vaccine system under ultraviolet light for 30 seconds to obtain the hydrogel vaccine.

[0032] The wavelength of the ultraviolet light of the present invention is preferably 405nm, and the power is preferably 1W / cm 2 Rheological tests confirmed that the solution could form gel efficiently and release GM-CSF slowly.

[0033] The present invention also provides a hydrogel vaccine prepared by the above preparation method.

[0034] The present invention also provides the use of the hydrogel vaccine system or the hydrogel vaccine in the preparation of tumor immunotherapy drugs. When the hydrogel system is used in the present invention, the hydrogel vaccine system can be directly injected.

[0035] To further illustrate the present invention, a hydrogel vaccine system, hydrogel vaccine, preparation method and application of tumor immunotherapy provided by the present invention are described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0036] Example 1

[0037] (1) Preparation of senescent tumor cell membrane vesicles

[0038] Oxidative stress induced senescence of CT26.WT cells, and then the cells were collected for future use.

[0039] (2)GM-CSF+CNV R848 Preparation of @nanoGel smart hydrogel

[0040] Dissolve 0.1g GelMA in 1mL PBS containing initiator, dissolve in a water bath at 60℃~70℃ in dark for 20~30min, add 0.06g Nanoclay, and incubate at 50℃ in dark for 2h. When the temperature of GelMA / Nanoclay cools to room temperature in dark, add 80ng GM-CSF and 300μg senescent tumor cell membrane vesicles loaded with 10μg R848, shake evenly, and then irradiate the hydrogel pre-liquid at 405nm (1W / cm 2 ) wavelength light for 30s to form a hydrogel; rheological tests confirmed that this scheme can form a hydrogel efficiently and can release GM-CSF slowly.

[0041] (3) Disinfect the injection area with 75% alcohol cotton balls and inject 1×106 CT26.WT cells were used to construct a subcutaneous tumor-bearing model of CT26.WT mice with colon cancer.

[0042] (4) Vaccination (therapeutic): 7 days after tumor bearing, the hydrogel pre-liquid (hydrogel vaccine without light curing) was injected subcutaneously near the tumor and the photosensitive surface was illuminated at 405nm (1W / cm 2 ) wavelength light for 30s, which can effectively induce the formation of hydrogel in vivo;

[0043] (5) Analysis of the efficiency of senescent cell clearance in tumor tissues by flow cytometry and immunofluorescence Figure 2 ), T cell activation ( Figure 3 ), qRT-PCR was used to detect the expression of pro-inflammatory cytokine mRNA ( Figure 4 ). Record the tumor size of tumor-bearing mice ( Figure 5 ) and survival of tumor-bearing mice ( Figure 6 ), HE was used to evaluate tumor-draining lymph nodes and lung tumor metastasis.

[0044] qRT-PCR primer sequences:

[0045] Il-2 (SEQ ID No. 1)5'-TGAGCAGGATGGAGAATTACAGG-3' (Forward);

[0046] Il-2 (SEQ ID No. 2)5'-GTCCAAGTTCATCTTCTAGGCAC-3' (Reverse);

[0047] Ifn-γ(SEQ ID No.3)5'-ATGAACGCTACACACTGCATC-3'(Forward);

[0048] Ifn-γ(SEQ ID No.4)5'-CCATCCTTTTTGCCAGTTCCTC-3'(Reverse);

[0049] Tnf-α(SEQ ID No.5)5'-CCCTCACACTCAGATCATCTTCT-3'(Forward);

[0050] Tnf-α (SEQ ID No. 6) 5'-GCTACGACGTGGGCTACAG-3' (Reverse).

[0051] The results are as follows Figure 2 to Figure 6 As shown, compared with the PBS group and the Ctrl group (nanoGel), the smart hydrogel vaccine Vaccine (GM-CSF+CNVR848 @nanoGel) can effectively activate tumor immunity, inhibit tumor growth and prolong survival.

[0052] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for constructing a hydrogel vaccine system for tumor immunotherapy for combined elimination of senescent cells and tumor cells, characterized in that: The following steps are involved: After GelMA was initiated and dissolved, it was mixed with Nanoclay and incubated at 50°C in the dark for 2 h to obtain GelMA / Nanoclay; Mixing GelMA / Nanoclay with DC cell chemotactic factors and / or activation factors, and senescent tumor cell membrane vesicles pre-loaded with vaccine adjuvants to obtain the hydrogel vaccine system; The DC cell chemokine and / or activation factor includes GM-CSF; The vaccine adjuvant includes R848 or STING agonist cGAMP; The tumor types that induce senescence include solid tumor cells; The method for preparing induced senescence tumor cells comprises inducing tumor cell senescence by using oxidative stress damage, squeezing the induced senescence tumor cells and collecting cell membranes to prepare vesicle-like cells.

2. A method for preparing a hydrogel vaccine for tumor immunotherapy, characterized in that: The method comprises irradiating the hydrogel vaccine system constructed by the construction method of claim 1 under ultraviolet light for 30 seconds to obtain the hydrogel vaccine.

3. The preparation method according to claim 2, characterized in that: The wavelength of the ultraviolet light is 405nm and the power is 1W / cm 2 .

4. A hydrogel vaccine prepared by the preparation method according to claim 2 or 3.

5. Use of the hydrogel vaccine system constructed by the construction method of claim 1 or the hydrogel vaccine of claim 4 in the preparation of tumor immunotherapy drugs.

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