Tumor nano vaccine and preparation method thereof

Abstract

The invention relates to a tumor nano vaccine and a preparation method thereof, and belongs to the technical field of biological medicines. The tumor nano vaccine comprises tumor cell lysate nano particles, the tumor cell lysate nano particles take acryloyl esters as monomers, a biodegradable cross-linking agent is used, and a polymer shell with nano thickness is synthesized in situ on the surface of protein or tumor specific antigen in tumor cell lysate through free radical polymerization reaction. The tumor cell lysate nanoparticles are formed. The tumor cell lysate nanoparticles can enhance the immunogenicity of tumor antigens and improve the immune response of an organism to tumors. The tumor nano vaccine provided by the invention enhances the stability of the tumor antigen, ensures that the tumor antigen can circulate in vivo for a long time, induces the organism to generate cellular immunity, activates T cells, and realizes the effect of treating and / or preventing tumors.

Description

[0001] Cross-reference

[0002] The present invention claims priority from an application filed in China with application number 2023116086285, application date November 28, 2023, and invention name “A tumor nanovaccine and preparation method thereof”. The above application is incorporated herein by reference in its entirety as a part of this application. Technical Field

[0003] The invention relates to a tumor nano vaccine and a preparation method thereof, and belongs to the technical field of biomedicine. Background Art

[0004] As a method of immunotherapy, tumor vaccines can trigger highly specific immune responses, activate the patient's own immune system through active immunization, and achieve specific killing of tumor cells.

[0005] The tumor vaccines that are currently being studied more include protein or peptide vaccines, DC vaccines, DNA vaccines, and mRNA vaccines. Among various vaccines, protein or peptide vaccines are the simplest from preparation to clinical application. However, most tumor vaccines are still in Phase I and Phase II clinical trials, and most of the very few vaccines that have entered Phase III clinical trials have not shown the expected efficacy. The reason may be factors of the vaccine itself on the one hand, and related to the research cases themselves on the other hand. Due to the diversity of tumor pathogenesis and clinical manifestations, even tumors of the same type in clinical practice have huge differences between different patients. The diversity of tumors and the differences between different patients have led to the disadvantage of unstable treatment effects of existing therapeutic vaccines, which is specifically manifested in that tumor therapeutic vaccines have good efficacy in very few individuals, but in most cases the efficacy is very poor or even completely ineffective, which seriously hinders the technical development and application of tumor therapeutic vaccines.

[0006] Due to the poor stability and low immunogenicity of a single tumor specific antigen (TSA), the immune efficiency of tumor vaccines with a single TSA is low, which limits its further clinical application. To this end, tumor vaccines containing multiple antigens have been developed for tumor immunotherapy. One strategy is to use tumor cell lysate (TCL) containing multiple TSAs as the antigen source for tumor vaccines. However, TCL has some defects as a tumor vaccine: 1) The antigen has poor stability and is easily degraded after injection in vivo; 2) The antigen presentation effect in vivo is weak, and it is difficult to induce a high level of anti-tumor immune response. How to enhance the antigen presentation of TCL in vivo, enhance the anti-tumor specific T cell response induced by the vaccine, and improve the immune activation and therapeutic effect of cancer vaccines are the core and difficult issues in vaccine research. Summary of the Invention

[0007] Previous studies have shown that TCL can induce the activation of various immune cells, such as T lymphocytes, dendritic cells, monocytes, etc. However, the simple TCL vaccine has low immunogenicity and can only stimulate the body to produce a low level of immune response. Therefore, a good TCL vaccine needs to be combined with a suitable adjuvant and modified to enhance the efficacy of the vaccine. In view of the problems of tumor immunotherapy, the present invention provides a tumor nano-vaccine and its preparation method. Specifically,

[0008] In the first aspect of the present invention, there is provided a tumor nano-vaccine, which comprises tumor cell lysate (TCL) and tumor cell lysate nanoparticles formed by acrylate polymer and crosslinking agent.

[0009] Preferably, the acrylate polymer is polymerized from one or more of acrylate monomers. More preferably, the acrylate monomers include cationic monomers.

[0010] Further preferably, the acrylate monomers include one or more of acrylamide (AAM), methacryloxyethyltrimethylammonium chloride (DMC), poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide, vinylpyrrolidone, acryloyloxyethyltrimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate, 3-[(3-acrylamidopropyl)dimethylammonium] propionate. More preferably, the polymer includes a copolymer formed by AAM and DMC.

[0011] Preferably, the crosslinking agent is a biodegradable crosslinking agent. More preferably, the crosslinking agent is triblock poly(lactic acid)-poly(ethylene glycol)-dimethylacrylate (AC-PLA-PEG-PLA-AC).

[0012] More preferably, the preparation method of the AC-PLA-PEG-PLA-AC includes:

[0013] (1) Synthesize PLA-PEG-PLA; (2) Synthesize AC-PLA-PEG-PLA-AC.

[0014] More preferably, in step (1), PEG and DL-lactide (DL-LA) are used as raw materials to polymerize into PLA-PEG-PLA. More preferably, the mass ratio of PEG to DL-LA is (1-5):1, which can be any range or value within the above range, such as (2-5):1, 2.5:1, 3:1, 4:1, etc.

[0015] More preferably, in step (2), acryloyl chloride is added for polymerization. More preferably, the mass-volume ratio of PLA-PEG-PLA to acryloyl chloride is (1-5):1 (g / ml), which can be any range or value within the above range, such as (2-5):1, 2.5:1, 3:1, 4:1, etc.

[0016] Preferably, the tumor cell lysate is derived from any tumor cells, including any tumor cells derived from the nervous system, digestive system, circulatory system, urinary system, endocrine system, and / or reproductive system.

[0017] In a specific embodiment, the tumor cell lysate is derived from tumor cells such as melanoma cells, breast cancer cells, colorectal cancer cells, ovarian cancer cells, osteosarcoma cells, pancreatic cancer cells, gastric cancer cells, lung cancer cells, chordoma cells, cervical cancer cells, nasopharyngeal cancer cells, thyroid cancer cells, bladder cancer cells, esophageal cancer cells, gallbladder cancer cells, prostate cancer cells, etc.

[0018] More preferably, the particle size range of the tumor cell lysate nanoparticles is 20-50 nm.

[0019] More preferably, the polymer and the cross-linking agent form a shell encapsulating the tumor cell lysate.

[0020] More preferably, the molar ratio of the antigen concentration of the tumor cell lysate, acrylate monomer, and cross-linking agent is 1:(4500-7500):(200-400), which can be any value within the above ratio, such as 1:4500:400, 1:7500:200, 1:5000:300, 1:6000:300, 1:7000:270, etc.

[0021] More preferably, the monomers include AAM and DMC, and the molar ratio of monomer AAM:DMC is (6-12):1, which can be any value within the above ratio, such as 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, etc.

[0022] In a specific embodiment, the molar ratio of the antigen concentration of the tumor cell lysate:AAM:DMC:cross-linking agent is 1:5400:600:400.

[0023] Preferably, the antigen of the tumor cell lysate in a single dose of the tumor nano-vaccine is 5 μg - 10 mg.

[0024] Preferably, the tumor nano-vaccine further comprises an adjuvant.

[0025] More preferably, the adjuvant comprises a substance that can stimulate the body to produce a stronger humoral and / or cellular immune response against the antigen co-administered therewith. The adjuvants described herein may be well-known to those skilled in the art and include, but are not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponins, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic component adjuvants (such as calcium adjuvants), cytokine and nucleic acid adjuvants (such as monocyte colony-stimulating factor, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid carriers, etc.), MPL-A adjuvant, emulsion adjuvants (such as Freund's adjuvant). The adjuvant may be a pharmaceutically acceptable adjuvant.

[0026] In one or more embodiments of the present invention, the adjuvant is a CpG adjuvant and / or an MPL-A adjuvant.

[0027] Preferably, the mass ratio of the tumor cell lysate nanoparticles to the adjuvant is (2 - 8):(1 - 3), which can be any range or any value within the above range, such as 2; 3, 2:2,, 2:1, 3:1, 5:3, 6:1, 8:1, 8:3, etc.

[0028] In a specific embodiment, the adjuvant is a CpG adjuvant and an MPL-A adjuvant, and the mass ratio of CpG to MPL-A is (1 - 3):1, which can be any range or any value within the above range, such as 1:1, 2:1, 3:1, etc.

[0029] Preferably, the tumor nano-vaccine up-regulates macrophage and dendritic cell subsets cDC1 and cDC2.

[0030] Preferably, the tumor nano-vaccine up-regulates the proportion of T cell responses. More preferably, the T cells include activated (CD69+) T cells and / or memory T cells.

[0031] In a second aspect of the present invention, there is provided a method for preparing any of the above tumor nano-vaccines, the preparation method comprising:

[0032] (1) Preparing a tumor cell lysate;

[0033] (2) Dissolving acrylate monomers in a buffer at room temperature;

[0034] (3) Mix the tumor cell lysate (TCL), acrylate monomer, crosslinker, initiator, and buffer at room temperature;

[0035] (4) Conduct a polymerization reaction at 4 °C to form tumor cell lysate nanoparticles;

[0036] (5) Dialyze the obtained reaction product to obtain tumor cell lysate nanoparticles n(TCL).

[0037] Preferably, step (1) of the preparation method includes:

[0038] (1-1) Incubate the tumor tissue with collagenase to prepare a single-cell suspension;

[0039] (1-2) Wash the single-cell suspension with buffer to prepare a cell suspension;

[0040] (1-3) Perform multiple freeze-thaw cycles on the cell suspension in a -37 °C freezing water bath to break the cells and obtain a broken cell mixture;

[0041] (1-4) Centrifuge and filter the broken cell mixture to obtain the tumor cell lysate.

[0042] More preferably, the multiple freeze-thaw cycles include 4-8 times, and further preferably 4, 5, 6, 7, or 8 times.

[0043] Preferably, in step (2),

[0044] The acrylate monomer includes acrylamide (AAM), methacryloxyethyltrimethylammonium chloride (DMC), poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine, N-(3-aminopropyl) methacrylamide, vinylpyrrolidone, acryloyloxyethyltrimethylammonium chloride,

[0045] [2-(Methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate, 3-[(3-acrylamidopropyl)dimethylammonium] propionate, or one or more of them.

[0046] Preferably, in step (3),

[0047] The antigen concentration of the tumor cell lysate, the molar ratio of the monomer to the crosslinking agent is 1:(4500 - 7500):(200 - 400), which can be any value within the above ratio, such as 1:4500:400, 1:7500:200, 1:5000:300, 1:6000:300, 1:7000:270, etc. The antigen concentration is calculated based on N in the tumor cell lysate.

[0048] More preferably, the monomer is AAM and DMC. Further preferably, the molar ratio of monomer AAM:DMC is (6 - 12):1, which can be any value within the above ratio, such as 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, etc.

[0049] In a specific embodiment, the molar ratio of the antigen concentration of the tumor cell lysate:AAM:DMC:crosslinking agent is 1:5400:600:400.

[0050] More preferably, the polymer comprises a copolymer formed by AAM and DMC.

[0051] Preferably, the crosslinking agent is a biodegradable crosslinking agent. More preferably, the crosslinking agent is triblock poly(lactic acid)-poly(ethylene glycol)-dimethacrylate (AC-PLA-PEG-PLA-AC).

[0052] Preferably, the tumor cell lysate is derived from tumor cells such as melanoma cells, breast cancer cells, colorectal cancer cells, ovarian cancer cells, osteosarcoma cells, pancreatic cancer cells, gastric cancer cells, lung cancer cells, chordoma cells, cervical cancer cells, nasopharyngeal cancer cells, thyroid cancer cells, bladder cancer cells, esophageal cancer cells, gallbladder cancer cells, prostate cancer cells, etc.

[0053] Preferably, the molar ratio of the protein of the tumor cell lysate to the initiator is 1:(200 - 400). It can be any range or any value within the above range, such as 1:200, 1:250, 1:300, 1:350, 1:400, etc.

[0054] More preferably, the initiator includes one or more of ammonium persulfate (APS), sodium persulfate, and potassium persulfate.

[0055] More preferably, step (3) further includes adding a catalyst, and the molar ratio of the protein of the tumor cell lysate to the catalyst is 1:

[0056] (500 - 700). It can be any range or any value within the above range, such as 1:500, 1:550, 1:600, 1:650, 1:700, etc.

[0057] Further preferably, the catalyst includes tetramethylethylenediamine (TEMED).

[0058] Preferably, the buffer solution includes phosphate buffer solution.

[0059] Preferably, the tumor cell lysate contains 50 - 500 μg / 10 6 cells of protein (antigen).

[0060] Preferably, the polymerization reaction in step (4) includes: initiating a radical polymerization reaction on the surface of the tumor antigen to encapsulate the tumor cell lysate (TCL) within a shell formed by a polymer and a crosslinking agent, thereby forming tumor cell lysate nanoparticles.

[0061] More preferably, the particle size range of the tumor cell lysate nanoparticles is 20 - 50 nm.

[0062] Preferably, the preparation method further includes (6) purifying the tumor cell lysate nanoparticles n(TCL).

[0063] In the third aspect of the present invention, there is provided the use of any one of the above - mentioned tumor nano - vaccines in the preparation of a pharmaceutical composition for treating and / or preventing tumors.

[0064] Preferably, the tumor is one of melanoma, breast cancer, colorectal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, gastric cancer, lung cancer, chordoma, cervical cancer, nasopharyngeal cancer, thyroid cancer, bladder cancer, esophageal cancer, gallbladder cancer, prostate cancer, etc.

[0065] In the fourth aspect of the present invention, there is provided a pharmaceutical composition, which includes any one of the above - mentioned tumor nano - vaccines or tumor cell lysate nanoparticles.

[0066] Preferably, the pharmaceutical composition further includes pharmaceutically acceptable excipients.

[0067] More preferably, the excipients include osmotic pressure regulators and surfactants.

[0068] More preferably, the dosage form of the pharmaceutical composition is any one or several of liquid preparations, powders, injections, and tablets; further preferably, the pharmaceutical composition is a subcutaneous injection, intramuscular injection, or microneedle injection.

[0069] In the fifth aspect of the present invention, there is provided a method for treating and / or preventing tumors, and the treatment method includes administering any one of the above - mentioned tumor nano - vaccines or any one of the above - mentioned pharmaceutical compositions to a subject.

[0070] Preferably, the tumor is one of melanoma, breast cancer, colorectal cancer, ovarian cancer, osteosarcoma, pancreatic cancer, gastric cancer, lung cancer, chordoma, cervical cancer, nasopharyngeal cancer, thyroid cancer, bladder cancer, esophageal cancer, gallbladder cancer, prostate cancer, etc.

[0071] Preferably, the subject can be a human or a non-human animal.

[0072] Furthermore, the non-human animal can be a non-human mammal.

[0073] The non-human mammal can be any one of mice, rats, guinea pigs, hamsters, pigs, dogs, sheep, monkeys, rabbits, cats, cows, horses, etc., but is not limited thereto.

[0074] The administration described herein includes but is not limited to intramuscular injection, subcutaneous injection, intradermal injection, intravenous injection, arterial injection, intratumoral injection, intraperitoneal injection, microneedle injection, mucosal administration, oral administration, oral and nasal spraying, or aerosol inhalation.

[0075] Advantageous technical effects of the present invention:

[0076] 1. The raw materials of the tumor nano-vaccine of the present invention are simple in source and easy to obtain. After lysing tumor cells, antibodies capable of inducing cellular immunity in the body can be obtained;

[0077] 2. In the preferred embodiment, in the tumor nano-vaccine of the present invention and its preparation method, the monomer DMC is a cationic monomer. Due to the negatively charged characteristics of the antigens in the tumor cell lysate, adding a positively charged monomer (DMC) can improve the encapsulation rate;

[0078] 3. The tumor nano-vaccine of the present invention uses a biodegradable cross-linking agent. The degradation of the cross-linking agent causes the degradation of the vaccine shell, thereby releasing antigens and activating cellular immunity in the body.

[0079] 4. The present invention adopts in-situ free radical polymerization. Monomers and cross-linking agents are enriched around antigens through hydrogen bonding and electrostatic adsorption, forming a cross-linked network to encapsulate the antigens, and preparing a tumor antigen nano-vaccine. This tumor vaccine can enhance the immunogenicity of tumor antigens, improve the immune response of the body to tumors. The tumor nano-vaccine of the present invention enhances the stability of tumor antigens, ensures their long-term circulation in the body and activates cellular immunity of the body, effectively inhibiting the growth of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 It is a flowchart for the extraction of tumor cell lysate.

[0081] Figure 2 It is a TEM characterization diagram of the tumor nano-vaccine, where Figure A is n(TCL) and Figure B is n(OVA).

[0082] Figure 3 Schematic diagram of the process of immunizing mice with tumor nano-vaccine.

[0083] Figure 4 Shows the effect of tumor nano-vaccine n(TCL) in murine skin cancer B16F10. The left figure is the tumor growth curve, and the right figure is the survival curve.

[0084] Figure 5 Qualitative analysis of related immune cells after the action of tumor nano-vaccine n(OVA).

[0085] Figure 6 Quantitative analysis of related immune cells in draining lymph nodes and non-draining lymph nodes after the action of tumor nano-vaccine n(OVA).

[0086] Figure 7 Response analysis of T cells at the draining lymph nodes after the action of tumor nano-vaccine n(OVA).

[0087] Figure 8 Synthesis schematic diagram of PLA-PEG-PLA.

[0088] Figure 9 Synthesis schematic diagram of AC-PLA-PEG-PLA-AC.

[0089] Figure 10 1H NMR spectrum of PLA-PEG-PLA. a, b, c, d, e are the 1H NMR spectra of PLA-PEG-PLA at 5.21, 6.29, 7.18, 7.15-1, 7.15-2 respectively.

[0090] Figure 11 Mass spectrometry results of PLA-PEG-PLA.

[0091] Figure 12 1H NMR spectrum of AC-PLA-PEG-PLA-AC. a, b, c, d, e are the 1H NMR spectra of AI102 at 5.21, 6.29, 7.18, 7.15-1, 7.15-2 respectively.

[0092] Figure 13 Mass spectrometry results of AC-PLA-PEG-PLA-AC. a, b, c, d, e, f, g are the mass spectrometry results of AI102 at 5.21, 6.29, 7.18, 7.15-1, 7.15-2. Specific implementation manners

[0093] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not constitute any limitation to the present invention in any way.

[0094] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0095] Example 1 Preparation of Tumor Nano-Vaccine

[0096] 1.1 Extraction of Tumor Cell Lysates

[0097] (1) Inject 2×10 6 B16F10 cells into the right subcutaneous area of the back of C57BL / 6 to prepare B16F10 (melanoma) tumor model mice. Collect tumor tissues from the mice on the 13th day after tumor implantation.

[0098] (2) Incubate the tumor tissues with collagenase (1 mg / ml) for 30 min and then collect the single-cell suspension through a 70-μm cell sieve.

[0099] (3) Wash the single-cell suspension collected in (2) with cold PBS and resuspend it in PBS at a density of 10 7 cells / mL.

[0100] (4) Subject the washed cell suspension in (3) to 5 freeze-thaw cycles in liquid nitrogen and a 37°C water bath to break the cells, then centrifuge the obtained mixture at 1000×g for 10 min, collect the supernatant and filter it through a 0.22-μm sterile filter membrane to obtain tumor cell lysates (TCL).

[0101] (5) Use the BCA assay method to measure the protein concentration in the supernatant, with bovine serum albumin as the standard, and obtain tumor cell lysates with a protein (antigen) content of 100 - 300 μg / 10 6 cells.

[0102] 1.2 Preparation of Tumor Nano-Vaccine

[0103] (1) Collect the tumor cell lysate with a protein concentration of 1 - 3 mg / mL in an EP tube. Then, successively add 1×PBS, TCL, AAM, DMC, and AC-PLA-PEG-PLA-AC to the EP tube according to the molar ratio of protein concentration in the tumor cell lysate (TCL): acrylamide (AAM): methacryloyloxyethyl trimethyl ammonium chloride (DMC): triblock poly(lactic acid)-poly(ethylene glycol)-diacrylate (AC-PLA-PEG-PLA-AC) of 1:5400:600:400. After adding, vortex to mix the system evenly. Subsequently, add ammonium persulfate (APS) to the test tube according to the ratio of TCL:APS = 1:300. After adding, vortex again to mix evenly. Then, add N,N,N',N'-tetramethylethylenediamine (TEMED) to the test tube according to the ratio of TCL:TEMED = 1:600. React at 4°C for 2 h through free radical polymerization reaction.

[0104] (2) Purification of n(TCL): Unreacted n(TCL) is removed using an ultrafiltration membrane with a molecular weight cut-off of 100 kDa, and the washing buffer is 10 mM phosphate buffer solution (PBS, pH 7.2 - 7.4). Then, the obtained tumor cell lysate nanoparticles n(TCL) are stored at 4°C for subsequent experimental use.

[0105] The process schematic diagram is as Figure 1 shown.

[0106] The obtained n(TCL) was observed under a transmission electron microscope (TEM), as Figure 2 shown. The left Figure 2 Panel A is the morphological characterization of n(TCL). It can be seen that n(TCL) is spherical, with a size of about 20 nm and a uniform distribution.

[0107] Example 2 Functional Evaluation of Tumor Nanovaccine

[0108] 2.1 Effect of Nanovaccine on the Growth of Mouse Melanoma

[0109] (1) On day 0, inject 2×10 6 B16F10 cells into the right subcutaneous area of the back of C57BL / 6 to prepare B16F10 tumor model mice;

[0110] (2) On days 3, 5, and 10, inject the n(TCL) anti-tumor vaccine containing CpG and MPL-A adjuvant subcutaneously into the tumor model mice. The doses of n(TCL), CpG, and MPL-A injected into each mouse are 50 μg, 20 μg, and 10 μg, respectively. Use the PBS group as a negative control and compare it with the TCL group in which TCL replaces n(TCL);

[0111] (3) On the 80th and 130th days, 2×10 6 B16F10 cells were injected subcutaneously into the right side of the surviving tumor model mice treated with (TCL)n;

[0112] (4) From the 0th day to the 180th day, the tumor volume was measured every two days, and the survival rate of the mice was counted;

[0113] The process schematic diagram is as Figure 3 .

[0114] The results are as Figure 4 shown. As Figure 4 shown in the left figure, observing the size of the mice's tumors in the first 50 days, the tumors in the n(TCL) group hardly grew, and the survival rate was 100%. In the PBS negative control group, the tumors rapidly reached 2000 mm 3 around the 20th day and all died on the 23rd day. In the TCL group, the tumors reached 1500 mm 3 around the 27th day and all died on the 47th day. Whether compared with the negative control group or the TCL group, there were significant differences in the tumor size and survival rate in the n(TCL) group.

[0115] As Figure 4 shown in the right figure, after re-injecting tumor cells into the surviving n(TCL) group on the 80th and 130th days, the survival rate was not affected and remained at 100%, and no tumor growth was observed.

[0116] As can be seen from Table 1: The mice inoculated with the anti-tumor vaccine did not show tumor growth, and the survival rate was 100%, while the tumor volume of the mice in the negative control group exceeded 1000 mm 3 , and all died, indicating that the n(TCL) anti-tumor vaccine containing CpG and MPL-A adjuvants can inhibit the growth of B16F10 tumor cells and has a tumor treatment effect.

[0117] Table 1 Tumor volume record form

[0118]

[0119]

[0120] 2.2 Immunological evaluation experiment of mice inoculated with nano-vaccine

[0121] Replace the TCL in Example 1 with the model tumor antigen ovalbumin (OVA) to obtain n(OVA) by the obtained method ( Figure 2 B).

[0122] (1) The model tumor antigen chicken ovalbumin (OVA) or n(OVA) plus CpG and MPL-A adjuvants were subcutaneously injected into BALB / c mice (refer to the ratio and method in Section 2.1);

[0123] (2) draining and nondraining lymph nodes were collected on day 7 for flow cytometric analysis;

[0124] (3) Soak the collected lymphoid tissue in 0.1% BSA / PBS (m / v) solution. Grind the above tissue into a homogenate using a tissue grinder and filter through a cell sieve.

[0125] (4) The homogenate is centrifuged and red blood cell lysis solution is added. After lysis is completed, an appropriate amount of BSA / PBS (m / v) solution is added, and the supernatant is discarded after centrifugation again. The above steps are repeated until the precipitate at the bottom is white, and the red blood cell lysis step is completed.

[0126] (5) After discarding the homogenate of tissue cells with complete red blood cell lysis, add an equal volume of BSA / PBS (m / v) solution (number of indicators / ml) according to the number of test indicators and pipette evenly. Then, prepare the corresponding number of 1.5 ml test tubes according to the number of indicators to be tested, and add 1 ml of the tissue cell homogenate pipette evenly to each test tube for later use.

[0127] (6) Place the 1.5 ml tube containing 1 ml of tissue cell homogenate in a refrigerated centrifuge and centrifuge at 3000 r / min for 3 min at 4°C. Discard the supernatant and add 100 μl of antibody pre-liquid (BV421-CD11b, FITC-CD11c, PE-CD103, AF700-F4 / 80, APC / CY7-CD45; APC / CY7-CD3, BV421-CD4, AF700-CD8, PE / CY7-CD69, PE-SIINFKEL). Incubate in the dark for 30 min and use a cell flow cytometer to detect relevant immune indicators.

[0128] The results are as follows Figures 5-7 As shown,

[0129] Figure 5 The results showed that we successfully used flow cytometry to distinguish different types of immune cells, including macrophages and dendritic cell subsets cDC1 and cDC2, which are very important for antigen presentation of cancer vaccines.

[0130] By flow cytometry analysis, Figure 6It is shown that both cDC1 and cDC2, the dendritic cell subsets in the draining lymph nodes, are significantly upregulated. These two are the DCs that promote T cell and B cell responses. Meanwhile, the OVA-specific T cells in the draining lymph nodes are also significantly upregulated statistically compared to those in the non-draining lymph nodes( Figure 7 ), indicating that the n(TCL) encapsulated in the housing of the present invention can effectively enhance the cellular immune response and improve the immunogenicity. For details, please refer to Tables 2 and 3.

[0131] Table 2 Counts of related immune cells in draining and non-draining lymph nodes after the action of tumor nano-vaccine n(OVA)

[0132]

[0133] Table 3 Response ratio of T cells at the draining lymph nodes after the action of tumor nano-vaccine n(OVA)

[0134]

[0135]

[0136] Example 3 Preparation and Characterization of Triblock Polylactic Acid Polyethylene Glycol Dimethacrylate (AC-PLA-PEG-PLA-AC)

[0137] The above crosslinking agent AC-PLA-PEG-PLA-AC can be prepared by the following method:

[0138] 1. Synthesis of PLA-PEG-PLA (see the synthesis schematic diagram in Figure 8 ):

[0139] (1) Dry the reaction vessel cap at 80 °C and the bottle at 120 °C for two hours (put in a small magnetic stir bar), and heat the oil bath to 100 °C

[0140] (2) Dehydration of PEG: Weigh 5 g of PEG, deoxygenate and dehydrate it. There is no need to pass argon gas. Evacuate for 2 h (two wells are required)

[0141] (3) Tighten the reaction vessel cap, take the reaction tube out of the oil bath, adjust the oil bath to 140 °C, and remove one well

[0142] (4) First, aspirate the vent tube of the reaction tube and the vent tube of stannous octoate three times. Keep argon gas flowing at the end. Take 42 μl of stannous octoate (0.615 mol / L) and add it to the reaction tube. Then take 2.16 g of DL-LA and add it to the reaction tube. After taking, deoxygenate and seal the tube with argon gas.

[0143] (5) After adding, react the reaction tube in the oil bath at 140 °C for 6 h.

[0144] (6) Purify with n - hexane and dichloromethane. Dissolve the sample in dichloromethane with a total volume of about 10 - 15 ml, and drop it into about 225 ml of n - hexane, and wash it 4 - 5 times repeatedly.

[0145] (7) Dry. Cover with filter paper and place it in the fume hood for 1 day, then vacuum dry it overnight for the next step of synthesis.

[0146] Synthesis of 2AC - PLA - PEG - PLA - AC (see the synthesis schematic diagram in Figure 9 ):

[0147] (1) Dry the constant - pressure dropping funnel and round - bottom flask at 120 °C for 2 h

[0148] (2) Open the argon gas and vacuum pump, use an ice bath, aspirate three times through the anhydrous dichloromethane gas pipe, aspirate for a longer time in the round - bottom flask and pass argon gas. Take about 70 ml of anhydrous dichloromethane in the round - bottom flask, and add triethylamine according to the ratio of 5 g:2.2 ml. Take another about 10 ml of anhydrous dichloromethane in a test tube, add acryloyl chloride into it according to the ratio of 5 g:1.7 ml, and directly add a very small amount of inhibitor (dip a little with a dropper), pour it into the dropping funnel, and drip at a speed of 2 - 3 drops per second for more than 30 minutes, and react for 9 - 12 h.

[0149] (3) After the reaction, rotary evaporate to 10 - 15 ml, purify it in the same way as in the synthesis step of PLA - PEG - PLA, and dry it in the same way.

[0150] 3. Characterization of PLA - PEG - PLA

[0151] Figure 10 a, b, c, d, e are the 1H - NMR spectra of different batches of PLA - PEG - PLA. By comparing with the 1H - NMR spectra of PEG1000, D, L - lactide and PLA - PEG - PLA, it is found that PLA - PEG - PLA has both the characteristic peaks of PEG and the characteristic peaks of DL - lactide, and there are also new characteristic peaks generated by the combination of the two (peak b: at 4.3 ppm, which is the characteristic hydrogen of the methylene PLA - COO - CH2 in PEG connected to PLA).

[0152] Calculate how many lactide half - rings are attached to each PEG 1000: S peg (The peak at 3.6 ppm) / S DL-丙交酯 (The peak at 5.2 ppm) = 84 / x, and the calculated ratios are found to be 1:3.5, 1:4.18, 1:3.61, 1:4.14, 1:3.83 respectively.

[0153] Figure 11a is the mass spectrometry result of PLA-PEG-PLA. Analysis found that the molecular weight of PLA-PEG-PLA is 1288. In the first step of the reaction, D,L-lactide was added in a ratio of 1:2 (the ring opening of lactide forms two half rings). The molecular weight of D,L-lactide is 144, which matches the NMR result.

[0154] 4. Characterization of AC-PLA-PEG-PLA-AC

[0155] Figure 12 a, b, c, d, e are the 1H NMR spectra of different batches of AC-PLA-PEG-PLA-AC respectively. Compared with the PLA-PEG-PLA triblock, it can be seen that three characteristic peaks a, b, and c are generated, which are the three hydrogens at both ends of the double bond respectively.

[0156] Calculate the number of double bonds connected: S peg (The peak at 3.6 ppm) / S DL-丙交酯 (The peak at 6.2 or 5.9 or 6.5 ppm) = 84 / x. Calculation found that their ratios are 1:1.4, 1:1.2, 1:1.31, 1:1.35, 1:1.32 respectively.

[0157] Figure 13 a, b, c, d, e, f, g are the mass spectrometry results of AC-PLA-PEG-PLA-AC synthesized at 5.21, 6.29, 7.18, 7.15-1, 7.15-2. Analysis found that the molecular weight is 1400. In the second step of the reaction, acryloyl chloride was added. The molecular weight of acryloyl chloride is 90.5, which also matches the NMR result.

[0158] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0159] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

[0160] In addition, any combination can be made between various different embodiments of the present invention as long as it does not violate the idea of the present invention, and it should also be regarded as the content disclosed by the present invention.

Claims

1. A tumor nanovaccine, characterized in that: The tumor nano-vaccine comprises tumor cell lysate (TCL), tumor cell lysate nano-particles formed by acrylic acid ester polymer and cross-linking agent.

2. The tumor nanovaccine according to claim 1, characterized in that The acrylic polymer is polymerized by one or more acrylic monomers. Preferably, the acrylic monomers include one or more of acrylamide (AAM), methacryloyloxyethyltrimethylammonium chloride (DMC), poly(ethylene glycol) methyl ether acrylate, 2-methacryloyloxyethyl phosphorylcholine (MPC), N-(3-aminopropyl) methacrylamide (APM), vinyl pyrrolidone, acryloyloxyethyltrimethylammonium chloride, [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propyl) ammonium hydroxide, 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate, and 3-[(3-acrylamidopropyl)dimethylammonium] propionate. More preferably, the polymer includes a copolymer formed by AAM and DMC.

3. The tumor nanovaccine according to any one of claims 1-2, characterized in that: The cross-linking agent is a biodegradable cross-linking agent. Preferably, the cross-linking agent is triblock polylactic acid polyethylene glycol dimethacrylate (AC-PLA-PEG-PLA-AC). More preferably, the molar ratio of the antigen concentration of the tumor cell lysate, the acrylate monomer and the cross-linking agent is 1: (4500-7500): (200-400). Further preferably, the monomers include AAM and DMC, and the molar ratio of the monomers AAM: DMC is (6-12):

1.

4. The tumor nanovaccine according to any one of claims 1 to 3, characterized in that: The tumor cell lysate is derived from any tumor cell, including any tumor cell derived from the nervous system, digestive system, circulatory system, urinary system, endocrine system and / or reproductive system. Preferably, the tumor cell lysate is derived from melanoma cells, breast cancer cells, colorectal cancer cells, ovarian cancer cells, osteosarcoma cells, pancreatic cancer cells, gastric cancer cells, lung cancer cells, chordoma cells, cervical cancer cells, nasopharyngeal cancer cells, thyroid cancer cells, bladder cancer cells, esophageal cancer cells, gallbladder cancer cells, prostate cancer cells and other tumor cells. More preferably, a single dose of the tumor nanovaccine contains 5 μg-10 mg of the antigen of the tumor cell lysate.

5. The tumor nanovaccine according to any one of claims 1 to 4, characterized in that: The tumor nanovaccine also includes an adjuvant. Preferably, the adjuvant includes but is not limited to: plant adjuvants (such as alkylamines, phenolic components, quinine, saponin, sesquiterpenes, proteins, polypeptides, polysaccharides, glycolipids, phytohemagglutinins, etc.), bacterial adjuvants (such as cholera toxin, Escherichia coli heat-labile toxin, bacterial lipopolysaccharide, etc.), aluminum adjuvants and other inorganic adjuvants (such as calcium adjuvants), cytokines and nucleic acid adjuvants (such as monocyte colony stimulating factor, leukocyte factors IL-1, IL-2, IL-4, IL-5, IL-6, IFN-γ, CpG motifs, nucleic acid vectors, etc.), MPL-A adjuvants, emulsion adjuvants (such as Freund's adjuvant), more preferably, the mass ratio of tumor cell lysate nanoparticles: adjuvant is (2-8): (1-3), and further preferably, the tumor nanovaccine upregulates the response ratio of macrophages, dendritic cell subsets cDC1 and cDC2 and / or T cells.

6. The method for preparing the tumor nanovaccine according to any one of claims 1 to 5, characterized in that: The preparation method comprises: (1) preparing tumor cell lysates; (2) dissolving the acrylic acid ester monomer in a buffer solution at room temperature; (3) mixing tumor cell lysate (TCL), acrylate monomer, cross-linking agent, initiator and buffer at room temperature; (4) performing a polymerization reaction at 4°C to form tumor cell lysate nanopolymers; (5) The obtained reaction product is dialyzed to obtain tumor cell lysate nanoparticles n (TCL).

7. The preparation method according to claim 6, characterized in that: The step (1) of the preparation method comprises: (1-1) Tumor tissue is co-incubated with collagenase to prepare single cell suspension; (1-2) Washing the single cell suspension with a buffer solution to prepare a cell suspension; (1-3) The cell suspension is subjected to multiple freeze-thaw cycles in a -37°C water bath to disrupt the cells and obtain a disrupted cell mixture; (1-4) The broken cell mixture is centrifuged and filtered to obtain tumor cell lysate.

8. The preparation method according to any one of claims 6-7, characterized in that: The protein:initiator molar ratio of the tumor cell lysate is 1:(200-400). Preferably, the initiator comprises one or more of ammonium persulfate (APS), sodium persulfate, and potassium persulfate. More preferably, step (3) further comprises adding a catalyst. The protein:catalyst molar ratio of the tumor cell lysate is 1:(200-400). (500-700), further preferably, the catalyst comprises tetramethylethylenediamine (TEMED).

9. Use of the tumor nanovaccine according to any one of claims 1 to 5 in the preparation of a pharmaceutical composition for treating and / or preventing tumors.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the tumor nanovaccine according to any one of claims 1 to 5, and preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

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