CN115645520A

CN115645520A - Preparation method and application of chemotherapy-induced tumor protein antigen nano vaccine

Info

Publication number: CN115645520A
Application number: CN202211429002.3A
Authority: CN
Inventors: 闵元增; 马小鹏; 李�瑞; 郝玉浩
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2022-11-15
Filing date: 2022-11-15
Publication date: 2023-01-31

Abstract

The invention provides a preparation method and application of a chemotherapy-induced tumor protein antigen nano vaccine. The tumor protein antigen nano vaccine induced by chemotherapy, which is prepared by the method of the invention, has protein components with stronger antigenicity and adjuvant property, and can drain into lymph nodes by utilizing the size advantage, thereby realizing the effective delivery of tumor antigens and inducing organisms to generate anti-tumor immune response. The tumor protein antigen nano vaccine induced by chemotherapy can obviously improve the immunotherapy of tumors, provides a new idea for the development of tumor vaccines, and is easy for clinical transformation.

Description

Preparation method and application of chemotherapy-induced tumor protein antigen nano vaccine

Technical Field

The invention relates to the field of nano vaccines, in particular to a preparation method of a tumor protein antigen nano vaccine induced by chemotherapy and application of the tumor protein antigen nano vaccine in anti-tumor immunotherapy.

Background

Cancer is one of the public health problems that seriously threatens human life health. The latest statistical data of the international cancer research organization of the world health organization show that in 2020, the number of new cancer cases is 1929 ten thousand and the number of death cases is 996 ten thousand. The nobel physiology or medicine awards tumor immunotherapy in 2018, and then the anti PD-1 is widely applied to clinic and shows a good treatment effect in some tumor patients (such as melanoma patients). However, the anti PD-1 has the prominent disadvantage of low response rate and only plays a good treatment effect in a small part of tumor patients. Therefore, a plurality of combination treatment schemes combining chemotherapy, radiotherapy, targeted therapy and other means with immunotherapy appear clinically, and the synergistic treatment effect is expected to be achieved.

In numerous clinical trials, the combination of chemotherapy and immunotherapy is the most abundant, and shows great potential for tumor therapy. However, in many clinical trials, we have found that chemotherapy and immunotherapy do not always produce a positive synergistic effect. For example, a study published in Nature-medicine indicates that the combination of doxorubicin and antiPD-1 enhances the response rate of the single anti-iPD-1, while the therapeutic effect of the combination of cyclophosphamide and anti-iPD-1 is the opposite. To address this problem, the mechanism of action of chemotherapy needs to be followed. The effects of chemotherapy on immunotherapy can be divided into two parts. The first part is the direct effect of chemotherapy on tumor cells. The chemotherapy medicine directly kills tumor cells and releases tumor-associated antigen TAA and tumor neogenesis antigen TSA. These antigens are considered foreign substances and can induce mechanisms to generate anti-tumor immune responses. This is a manifestation of increased tumor antigenicity induced by chemotherapy. Meanwhile, the chemotherapy drugs induce tumor cells to generate immune stress and release a large amount of DAMPs related to damage molecules, and the DAMPs can be used as danger signals to activate and activate DC cells, so that the effect of enhancing immune response is achieved. This is a chemotherapy-induced increase in tumor antigenicity; the second component is that chemotherapy exhibits undifferentiated cytotoxicity, which is uncontrollable. Chemotherapeutic drugs can kill immunosuppressive cells such as BMDC, tregs, which are favorable for the anti-tumor immune response of the immune system. However, chemotherapeutic drugs can also kill T cells, DC cells, that exert an immune response, which can compromise the immune response of the body by tumor antigens. Thus, undifferentiated cytotoxicity may be the reason why chemotherapy in combination with immunotherapy does not always exert a synergistic therapeutic effect in certain clinical trials.

Disclosure of Invention

Based on the problems in the prior art, the invention provides a preparation method of a tumor protein antigen nano vaccine induced by chemotherapy and application thereof in anti-tumor immunotherapy, and aims to solve the problem that the combined use of chemotherapy and immunotherapy cannot exert a synergistic treatment effect. The vaccine preparation does not contain any carrier, is a whole-cell biological preparation, has better safety and biocompatibility, and is beneficial to clinical transformation and realization of personalized treatment.

The preparation method of the tumor protein antigen nano vaccine induced by chemotherapy takes damaged tumor cells, dead tumor cells and cell culture supernatant fluid treated by chemotherapy as a tumor antigen source, and prepares effective protein components (DAMPs, TAA, TSA and the like) in the tumor antigen into the nano vaccine with uniform particle size under the action of an organic solvent.

The chemotherapeutic agent includes, but is not limited to, oxaliplatin, cisplatin, carboplatin, paclitaxel, doxorubicin, cyclophosphamide, lobaplatin, nedaplatin, etoposide, irinotecan, pirarubicin, nimustine, carmustine, lomustine, ifosfamide, capecitabine, ganciclovir, deoxyfluoroguanosine, doxycycline, 5-fluorouracil, mercaptopurine, thioguanine, fluoroguanosine, tegafur, gemcitabine, carmofur, hydroxyurea, methotrexate, idodine, ancitabine, actinomycin D, daunorubicin, epirubicin, mitomycin, pellomycin, pingamycin, pirarubicin, cephalotaxine, hydroxycamptothecin, vinorelbine, taxotere, topotecan, vincristine, vindesine, elenine, etoposide, limonene, atamestane, anastrozole, amiloride, formestane, megestrol, captopril, altretamine, doxorubine, cabazine, cabazitaxel, doxylamine, or a combination of one or more thereof.

The organic solvent includes, but is not limited to, acetonitrile, ethanol, acetone, cyclohexane, acetaldehyde, acetamide, acetophenone, acrolein, acrylic acid, acrylonitrile, 3-chloropropene, aniline, trichlorotoluene, benzyl chloride, bischloromethyl ether, tribromomethane, carbon disulfide, carbon tetrachloride, benzene, xylene, toluene, trichloroethylene, trichloromethane, chlorobenzene, 2-chloro-1,3-butadiene, cumene, dimethyl sulfate, epichlorohydrin, ethyl acrylate, ethylbenzene, 1,2-dibromoethane, ethylene glycol, n-hexane, isophorone, methanol, dichloromethane, nitrobenzene, o-xylene, tetrachloroethylene, diethyl ether, ethyl acetate, ethyl acetoacetate, formic acid, isobutyraldehyde, methyl formate, n-butanol.

The tumor cell lines of the present invention include, but are not limited to, murine or human cell lines CT26, 4T1, LLC, B16F10, MC38, atT-20, B16, bEnd.3, beta-TC-6, C127, C6, FO, EL4, 9L/lacZ, hepa 1-6, MLTC-1, neuro-2a, L1210, OKT 11, P19, P388D1, P3X63Ag8, P3X63Ag8.653, P815, RG2, RAG, RH-35, RM-1, RP1846, MLTC-1, S-180, SHZ-88, SP2/0, sp2/0-Ag14, UMR-106, Y1, Y3-Ag 1.2.3, 35.1, YAC-1, 9L/lacZ, A-3, A172, 67375, BT-1080, daou-1080, daud H549, daud H-32, koeh 549, koeh-32, koeh MDA-MB-435S, MG-63, MM.1R, MM.1S, NCI-H2452[ H2452], RD, RT4, saos-2, SH-SY5Y, hela, A549, SK-N-SH, SW 1353, U-118MG, U-87MG, U-937, U251, WERI-Rb-1, 5637, 769-P, 786-O [786-0], 8305C, A-431, A-427, ACHN, HN AsPC-1, B-CPAP, C-33A, ca Ski, caco-2, calu-1, calu-3, calu-6, CAL-62, CFPAC-1, chago-K-1, COLO 205, COLO 320DM, CW-2, DLD-1, DMS 114, DU 145, ES-2, faDu, GBC-SD, HCC1937, HEC-1-A, HCT-15, HEC-1-A, hep 3B2.1-7, and, HT-29, HGC-27, hs 578T, huH-7, J82, JEG-3, KYSE-150, li-7, LNCaP clone FGC, loVo, LS 174T, MCF, MDA-MB-231, ME-180, MIAPaCa-2, MS751, NCCIT, NIH: OVCAR-3, PANC-1, PC-3, PC-9, siHa, RKO, SHP-77, SK-BR-3, T-47D, SW-13, SW1116, SW620, T-47D, T, T84, TCCSCST, UP-1, TT, UM-UC-3, VCaP, or ZR-75-1.

Tumors of the present invention include, but are not limited to, colorectal cancer, lung cancer, breast cancer, malignant melanoma, brain tumor, brain glioma, head and neck tumor, thyroid cancer, larynx cancer, oral breast cancer, cervical cancer, ovarian cancer, endometrial cancer, vulva cancer, esophageal cancer, cardia cancer, gastric cancer, liver cancer, large intestine cancer, rectal cancer, hepatic duct cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, papillary cancer, transitional epithelium cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, osteosarcoma, chondrosarcoma, malignant lymphoma, leukemia, choriocarcinoma, malignant hydatidiform mole, seminoma, germ cell tumor, intraocular melanoma, pregnant maroma, kidney cancer, skin cancer, malignant teratoma, malignant mesothelioma, retinoblastoma, non-hodgkin lymphoma, pancreatic cancer, penile cancer, pituitary tumor, non-small cell lung cancer, metastatic cancer, i.e., multiple endocrine adenoma, neuroblastoma, pancreatic tumor, kaposi tumor, primary and peritoneal tumor, primary small intestine tumor, urinary tract cancer, peritoneal sarcoma, etc.

The preparation method of the tumor protein antigen nano vaccine induced by chemotherapy comprises the following steps:

s1: the tumor cells are co-cultured with 1-300. Mu.M chemotherapeutic drug for 1-4 days (the tumor cells are cultured in flasks at a concentration of 60-200 ten thousand cells per ml), and the damaged tumor cells and the cell culture fluid are collected separately (the damaged cells, dead cells and cell culture supernatant are separated by centrifugation at 200-20000rpm for 5-60 minutes).

S2: freezing and thawing damaged tumor cells and dead tumor cells in liquid nitrogen and 37 ℃ water bath for 2-10 times, centrifuging at 200-20000rpm for 5-60min, collecting cell lysate, and combining with the cell culture solution obtained in step 1.

S3: and concentrating the mixed solution after S2 combination by using an ultrafiltration cup with the molecular weight cutoff of 1-100kDa, washing by using deionized water without endotoxin, and quantifying by using a Bradford protein detection kit to obtain a protein solution with the concentration of 0.1-10mg/mL.

S4: dropwise adding an organic solvent into a protein solution with the concentration of 0.1-10mg/mL at the speed of 0.1-9mL/min, wherein the volume ratio of the organic solvent to the protein solution is 1-20:1.

s5: stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning.

The tumor protein antigen nano vaccine induced by chemotherapy is a naked antigen cluster nanosphere, and an electron microscope photo of the tumor protein antigen nano vaccine is shown as a picture in figure 2.

The application of the tumor protein antigen nano vaccine induced by chemotherapy is to be used as a therapeutic nano vaccine pharmaceutical preparation in the process of tumor treatment.

The application of the tumor protein antigen nano vaccine induced by chemotherapy is that the nano vaccine is used as a therapeutic nano vaccine pharmaceutical preparation and is used together with immunotherapy in the process of chemotherapy and immunotherapy to play a synergistic treatment effect.

The invention has the beneficial effects that:

the tumor protein antigen nano vaccine induced by chemotherapy has protein components with stronger antigenicity and adjuvanticity, and drains into lymph nodes by utilizing the advantage of self size, thereby realizing the effective delivery of the tumor antigen. After DC cells mature, tumor antigens are presented to T cells, which in turn induce the body to mount an anti-tumor immune response. At the same time, immune checkpoint inhibitors are administered for the rescue of cytotoxic T lymphocytes in the tumor suppression microenvironment. The nano vaccine and the immunotherapy are combined for use, so that the response rate of the immunotherapy is improved. The nano vaccine has low preparation cost, and is favorable for clinical transformation and realization of personalized treatment.

Drawings

Figure 1 is the embodiment 2 chemotherapy induced tumor protein antigen nano vaccine preparation schematic diagram.

Figure 2 is a transmission electron micrograph (figure 2 a) and hydrodynamic diameter (figure 2 b) of the nano-vaccine of example 3.

FIG. 3 is the protein species analysis (FIG. 3a, c) and DMPAs analysis (FIG. 3 b) of the nano-vaccine prepared from example 4 cisplatin and oxaliplatin treatment of CT26 colorectal cancer cells.

Fig. 4 is the dosing schedule of the nano-vaccine in combination with immune checkpoint inhibitors (fig. 4 a), tumor growth curve (fig. 4b, c) and mouse survival curve (fig. 4 d) in the colorectal cancer model of example 5.

Fig. 5 is lymph node drainage (fig. 5a, b), DC cell capture (fig. 5 c) and induced DC cell maturation (fig. 5 d) of the example 6 nano-vaccine.

FIG. 6 is the in vitro spleen cell restimulation protocol for the nano-vaccine of example 7 (FIG. 6 a), flow cytometry and Elispot detection of interferon gamma (FIGS. 6 b-e).

FIG. 7 shows tumor T cell infiltration, flow cytometry detection (FIGS. 7 a-d) and immunohistochemistry (FIG. 7 e) induced by the NanoVan of example 8.

Detailed Description

In order to better illustrate the advantages and the innovations of the present invention, the present invention will be described with reference to the accompanying drawings and specific embodiments.

The invention relates to a preparation method of a tumor protein antigen nano vaccine induced by chemotherapy, which is characterized in that a tumor protein antigen generated by the induction of a chemotherapeutic drug is self-assembled under the action of an organic solvent to form the tumor protein antigen nano vaccine induced by chemotherapy.

The tumor cell lines of the present invention include, but are not limited to, murine or human cell lines CT26, 4T1, LLC, B16F10, MC38, atT-20, B16, bEnd.3, beta-TC-6, C127, C6, FO, EL4, 9L/lacZ, hepa 1-6, MLTC-1, neuro-2a, L1210, OKT 11, P19, P388D1, P3X63Ag8, P3X63Ag8.653, P815, RG2, RAG, RH-35, RM-1, RP1846, MLTC-1, S-180, SHZ-88, SP2/0, sp2/0-Ag14, UMR-106, Y1, Y3-Ag 1.2.3, 35.1, YAC-1, 9L/lacZ, A-3, A172, 67375, BT-1080, daou-1080, daud H549, daud H-32, koeh 549, koeh-32, koeh MDA-MB-435S, MG-63, MM.1R, MM.1S, NCI-H2452[ H2452], RD, RT4, saos-2, SH-SY5Y, hela, A549, SK-N-SH, SW 1353, U-118MG, U-87MG, U-937, U251, WERI-Rb-1, 5637, 769-P, 786-O [786-0], 8305C, A-431, A-427, ACHN, asPC-1, B-CPAP, C-33A, ca Ski, caco-2, calu-1, calu-3, calu-6, CAL-62, CFPAC-1, chago-K-1, COLO 205, COLO 320, CW-DM-2, DMS-1, HED 114, HE145, ES-2, FASD-357, GBSD-3532, HCC-357-C-34z32, HCC-35-H-1, HCC-3432, HCC-H-C-2, and HCC-B-C-2, HT-29, HGC-27, hs 578T, huH-7, J82, JEG-3, KYSE-150, li-7, LNCaP clone FGC, loVo, LS 174T, MCF, MDA-MB-231, ME-180, MIAPaCa-2, MS751, NCCIT, NIH: OVCAR-3, PANC-1, PC-3, PC-9, siHa, RKO, SHP-77, SK-BR-3, T-47D, SW-13, SW1116, SW620, T-47D, T, T84, TCCSCST, UP-1, TT, UM-UC-3, VCaP, or ZR-75-1.

Tumors in the present invention include, but are not limited to, colorectal cancer, lung cancer, breast cancer, malignant melanoma, brain tumor, brain glioma, head and neck tumor, thyroid cancer, larynx cancer, oral breast cancer, cervical cancer, ovarian cancer, endometrial cancer, vulval cancer, esophageal cancer, cardiac cancer, gastric cancer, liver cancer, large intestine cancer, rectal cancer, hepatic duct cancer, squamous cell cancer, basal cell cancer, adenocarcinoma, papillary cancer, transitional epithelial cancer, fibrosarcoma, malignant fibrous histiocytoma, liposarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, osteosarcoma, chondrosarcoma, malignant lymphoma, leukemia, choriocarcinoma, malignant hydatidiform mole, seminoma, germ cell tumor, intraocular melanoma, testicular cancer, kidney cancer, skin cancer, malignant teratoma, malignant mesothelioma, retinoblastoma, non-hodgkin lymphoma, pancreatic cancer, penile cancer, pituitary tumor, non-small cell lung cancer, metastatic cancer, i.e., multiple endocrine tumors, neuroblastoma, pancreatic tumors, nasopharyngeal tumors, peritoneal tumors, kaposi cancer, small intestine cancer, primary sarcoma, urethral cancer, and the like.

s1: the tumor cells are cultured in flasks at a concentration of 60-200 ten thousand cells per ml, and the tumor cells are co-cultured with the chemotherapeutic drug at a concentration of 1-300. Mu.M for 1-4 days. The damaged tumor cells, dead tumor cells and cell culture supernatant were collected from the chemotherapy treatment. Damaged cells, dead cells and cell culture supernatant were separated by centrifugation at 200-20000rpm for 5-60 minutes.

S3: and concentrating the cell lysate by using an ultrafiltration cup with the molecular weight cutoff of 1-100kDa, and washing by deionized water without endotoxin. Protein solution quantification the Bradford protein detection kit was used.

S4: the volume ratio of the organic solvent to the protein solution is 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min.

The tumor protein antigen nano vaccine induced by chemotherapy can be drained into lymph nodes by utilizing the advantage of self size, so that the effective delivery of the tumor antigen is realized. After DC cells mature, tumor antigens are presented to T cells, which in turn induce the body to mount an anti-tumor immune response. At the same time, immune checkpoint inhibitors are administered for the rescue of cytotoxic T lymphocytes in the tumor suppression microenvironment. The nano vaccine and the immunotherapy are combined to be used, so that the response rate of the immunotherapy is improved.

The present invention is further illustrated by the following specific embodiments. This example provides a preparation method of a chemotherapeutic induced tumor protein antigen nano vaccine for demonstration purposes only. The reagents used in the examples were all commercially available from Sigma, shandong platinum sources, pharmaceutical Co., ltd.

Example 1: extraction, purification and quantification of chemotherapy-induced tumor protein antigens

CT26 colorectal cancer-damaged cells, dead cells, and cell culture supernatant co-cultured with oxaliplatin at a concentration of 1-300. Mu.M or cisplatin at a concentration of 1-20. Mu.M were collected. Freeze thawing damaged cells and dead cells treated by chemotherapy in liquid nitrogen and 37 deg.C water bath for 2-10 times, centrifuging at 200-20000rpm for 5-60min, collecting cell lysate, and mixing with cell culture supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and washing with endotoxin-free deionized water. Protein quantification was performed using the Bradford protein detection kit.

Example 2: preparation of tumor protein antigen nano vaccine induced by chemotherapy

The volume ratio of the acetonitrile to the protein solution is 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min. Stirring, evaporating organic solvent, ultrafiltering with ultrafiltration tube with cut-off molecular weight of 1-100kDa (figure 1), and washing with deionized water without endotoxin.

Example 3: characterization of Nanoprotein vaccine

The nano-vaccine was dispersed in deionized water and transmission electron microscopy was taken after uranium acetate negative staining (fig. 2 a). The nano-vaccine was measured for potential and hydrodynamic diameter with a particle sizer (fig. 2 b).

Example 4: proteomics analysis of nano vaccine prepared by treating CT26 colorectal cancer cells with cisplatin and oxaliplatin

Proteins were collected and subjected to mass spectrometry including reduction, alkylation and trypsin digestion. To identify neoantigens, a library of mutated amino acid sequences was constructed that included 1-50 sites upstream and downstream of the mutation site, and only the detected sequences covering the mutation site were identified as neoantigens (FIGS. 3a, b, c). Compared with chemotherapy-induced DNA damage tumor cells, the protein vaccine has more concentrated immune response to the neutralizing epitope and can cause stronger immune response. This immune advantage is called immunodominance. Tumor specific antigen TSA, tumor associated antigen TAA and damage related molecular pattern DMPs in tumor cells are enriched by the tumor antigen protein nano vaccine induced by chemotherapy and verified by proteomics.

Example 5: pharmacodynamic test of nano vaccine combined immunotherapy prepared from oxaliplatin 2022/11/15 or cisplatin-treated tumor cells in CT26 colorectal cancer model

The method comprises the steps of implanting CT26 colorectal cancer cells into Balb/c mice subcutaneously, and injecting tumor nano vaccine and immune checkpoint blocker antiPD-1 at 5 days, 8 days and 11 days after tumor implantation. The dosage of the nano vaccine is 0.7mg per vaccine, the dosage of the anti PD-1 is 100 mu g per vaccine, and the administration is carried out for three treatment courses (figure 4 a). Observing and recording the body weight and the tumor volume of the mice when the tumor volume reaches 3000mm ³ At that time, the mice were sacrificed. The therapeutic effect is mainly reflected by the tumor volume and the mouse survival curve (fig. 4b, c, d).

The experimental results show that: the tumor protein antigen nano vaccine induced by chemotherapy, which is prepared by the method provided by the invention, is combined with the anti iPD-1, and compared with the single anti iPD-1 treatment, the tumor protein antigen nano vaccine induced by chemotherapy can improve the anti iPD-1 treatment effect and obviously prolong the survival time of a mouse.

Example 6: nano-vaccine drainage of lymph nodes and capture and maturation of DC cells

The near infrared fluorescent dye Cy5.5-NHS was used to prepare Cy5.5-labeled BSA by reacting with BSA at a charge ratio of 1. PBS, cy5.5-labeled BSA and BSA-NPs were injected subcutaneously into the left side of Balb/c mice. At 1 hour and 16 hours post-dose, mice were taken for left inguinal lymph nodes, imaged with IVIS and fluorescence intensity quantified, respectively (fig. 5a, b). Lymph nodes were ground and split red, flow-stained with live-dead and anti-mouse CD11c, CD80 and CD86 fluorescent antibodies for flow analysis of the capture of nano-vaccine and DC cell maturation by DC cells (fig. 5c, d).

The experimental results show that: the tumor protein antigen nano vaccine induced by chemotherapy, which is prepared by the method provided by the invention, can be effectively delivered to DC cells of lymph nodes and promotes more DC cells to mature.

Example 7: detection of interferon gamma secreted by spleen cells of mice by nano vaccine

One week after the last inoculation, spleens were isolated from Balb/c mice to obtain single cell suspensions. Splenocytes administered at 10 per well ⁵ The density of individual cells was seeded in ELISPOT-specific 96-well plates and co-incubated with 10-100. Mu.g of non-chemotherapy-induced tumor protein antigen or chemotherapy-induced tumor protein antigen. After 5-24 hours, biotinylated detection antibody, enzyme reagent and AEC substrate reagent were added according to the instructions for IFN- γ ELISPOT and the plates were read on the ELISPOT instrument (fig. 6a, b, c).

One week after the last inoculation, spleens were isolated from Balb/c mice to obtain single cell suspensions. Splenocytes were seeded in 12-well plates, 10 per well ⁶ The cells are then incubated with 10-100. Mu.g of non-chemotherapy-induced tumor protein antigen or chemotherapy-induced tumor protein antigen for 24-72 hours. Splenocytes were seeded at 4 ℃ using anti-mouse CD3, CD4 and CD8 flow antibodiesLine 5-30 min of staining. Finally, the cells were fixed, permeabilized and stained with anti-mouse IFN- γ antibody. Flow cytometry for detecting IFN gamma ⁺ CD8 ⁺ T cells and IFN-gamma ⁺ CD4 ⁺ Relative abundance of T cells (fig. 6a, d, e).

The experimental results show that: the tumor protein antigen nano vaccine induced by chemotherapy prepared by the method induces spleen cells of mice to secrete more interferon gamma in vitro and activates more CD8 secreting interferon gamma ⁺ T cells and CD4 ⁺ T cells.

Example 8: flow cytometry for analyzing infiltration of T lymphocytes at mouse tumor part

One week after the last dose, all mice were collected for tumor flow analysis. Mouse tumors were ground to single cell suspensions and lysed, and tumor cells were stained with vital dye at 4 ℃ for 5-30 min, followed by 5-30 min intracellular and extracellular antibody staining using anti-mouse CD16/CD32, CD45, CD3, CD4, CD8 and Foxp3 antibodies for flow analysis (fig. 7a, b, c, d).

One week after the last vaccination, tumor tissue was obtained from all treatment groups of mice. Tumor tissues were fixed with 4% paraformaldehyde for 12-48 hours and then dehydrated by embedding in paraffin. Next, tumors were sectioned at 4 μm and stained with anti-CD 3, CD4 and CD8 antibodies. Taken using tissue fax PLUS (fig. 7 e).

The experimental results show that: the tumor protein antigen nano vaccine induced by chemotherapy, which is prepared by the invention, can obviously improve the infiltration of cytotoxic T lymphocytes at tumor parts.

Example 9: extraction of human primary tumor cells to prepare tumor protein nano vaccine

Cutting tumor tissue to about 1mm ³ The pieces of tissue are washed 2-3 times with Hanks solution in a petri dish. 0.25% trypsin was added, shaken well and left overnight at 4 ℃. The next day, the mixture is washed with Hanks solution, the supernatant is discarded, and the washing is repeated for 2-3 times. Adding a small amount of culture medium containing serum, blowing, dispersing, counting cells, and culturing in separate bottles according to the concentration of 60-200 ten thousand cells per milliliter.

Damaged primary cells, dead primary cells, and cell supernatant were collected co-cultured with oxaliplatin at a concentration of 1-300. Mu.M or cisplatin at a concentration of 1-20. Mu.M. The tumor cells are frozen and thawed by liquid nitrogen and 37 ℃ water bath for 2-10 times and centrifuged for 5-60 minutes at 200-20000rpm, and then are combined with cell culture solution supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and washing. Taking acetonitrile and protein solution in a volume ratio of 1-20:1, acetonitrile is added dropwise at a rate of 1-9mL/min to a protein solution having a concentration of 1-10mg/mL. Stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning. Finally obtaining the tumor protein nano vaccine prepared by the human primary tumor cells.

Example 10: extracting primary tumor cells of patients who have not received chemotherapy to prepare tumor protein nano vaccine

Tumor tissue from colorectal cancer patients who have not received chemotherapy treatment is minced to about 1mm ³ The pieces of tissue are washed 2-3 times with Hanks solution in a petri dish. 0.25% trypsin was added, shaken well and left overnight at 4 ℃. The next day, the mixture is washed with Hanks solution, the supernatant is discarded, and the washing is repeated for 2-3 times. Adding a small amount of culture medium containing serum, blowing, dispersing, counting cells, and culturing in separate bottles according to the concentration of 60-200 ten thousand cells per milliliter.

Primary cells, dead primary cells and cell supernatant were collected from non-chemotherapy patients. The tumor cells are frozen and thawed by liquid nitrogen and 37 ℃ water bath for 2-10 times and centrifuged for 5-60 minutes at 200-20000rpm, and then are combined with cell culture solution supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and cleaning. Taking acetonitrile and protein solution in a volume ratio of 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min. Stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning. Finally obtaining the tumor protein nano vaccine prepared by the primary tumor cells of the patients without chemotherapy.

Example 11: extracting primary tumor cells of patients who have not received chemotherapy for drug treatment and preparing tumor protein nano vaccine

Tumors in colorectal cancer patients who have never received chemotherapyTumor tissue, which is minced to about 1mm ³ The pieces of crushed tissue were washed 2-3 times with Hanks solution in a petri dish. 0.25% trypsin was added, shaken well and left overnight at 4 ℃. The next day, the mixture is washed with Hanks solution, the supernatant is discarded, and the washing is repeated for 2-3 times. Adding a small amount of culture medium containing serum, blowing, dispersing, counting cells, and performing flask culture according to the concentration of 60-200 ten thousand cells per milliliter.

Oxaliplatin at a concentration of 1-300. Mu.M or cisplatin at a concentration of 1-20. Mu.M was added and co-cultured with primary cells from non-chemotherapeutic patients for 1-4 days. Damaged primary cells, dead primary cells and cell supernatant were collected from non-chemotherapy patients. The tumor cells are frozen and thawed by liquid nitrogen and 37 ℃ water bath for 2-10 times and centrifuged for 5-60 minutes at 200-20000rpm, and then are combined with cell culture solution supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and cleaning. Taking acetonitrile and protein solution in a volume ratio of 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min. Stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning. Finally obtaining the tumor protein nano vaccine cultured by the primary tumor cells in vitro by the medicament of the patient without chemotherapy.

Example 12: extracting primary tumor cells of patients who have undergone chemotherapy to prepare tumor protein nano vaccine

Tumor tissue from colorectal cancer patients treated with oxaliplatin or cisplatin is minced to about 1mm ³ The pieces of tissue are washed 2-3 times with Hanks solution in a petri dish. 0.25% trypsin was added, shaken well and left overnight at 4 ℃. The next day, the mixture is washed with Hanks solution, the supernatant is discarded, and the washing is repeated for 2-3 times. Adding a small amount of culture medium containing serum, blowing, dispersing, counting cells, and culturing in separate bottles according to the concentration of 60-200 ten thousand cells per milliliter.

Primary cells, dead primary cells and cell supernatant were collected from chemotherapy patients. The tumor cells are frozen and thawed by liquid nitrogen and 37 ℃ water bath for 2-10 times and centrifuged for 5-60 minutes at 200-20000rpm, and then are combined with cell culture solution supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and cleaning. Taking acetonitrile and protein solution in a volume ratio of 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min. Stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning. Finally obtaining the tumor protein nano vaccine prepared by the primary tumor cells of the chemotherapy patients.

Example 13: extracting primary tumor cells of patients who have undergone chemotherapy, culturing with in vitro medicine, and preparing tumor protein nano vaccine

Damaged primary cells, dead primary cells and cell supernatants from chemotherapy patients were collected co-cultured for 1-4 days with oxaliplatin at a concentration of 1-300. Mu.M or cisplatin at a concentration of 1-20. Mu.M. The tumor cells are frozen and thawed by liquid nitrogen and 37 ℃ water bath for 2-10 times and centrifuged for 5-60 minutes at 200-20000rpm, and then are combined with cell culture solution supernatant. Concentrating with ultrafiltration cup with molecular weight cutoff of 1-100kDa, and cleaning. Taking acetonitrile and protein solution in a volume ratio of 1-20:1, acetonitrile is added dropwise to a protein solution having a concentration of 1 to 10mg/mL at a rate of 1 to 9 mL/min. Stirring, after the organic solvent is volatilized, ultrafiltering the nano vaccine by using an ultrafiltration tube with the molecular weight cutoff of 1-100kDa, and cleaning. Finally obtaining the tumor protein nano vaccine cultured by the primary tumor cells in vitro medicaments of the chemotherapy patients.

The above-mentioned embodiments are intended to illustrate the objects, aspects and advantages of the present invention, and it should be understood that the above-mentioned embodiments are only examples of the present disclosure, and are not intended to limit the present disclosure, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present disclosure should be included in the scope of the present disclosure.

Claims

1. A preparation method of tumor protein antigen nano vaccine induced by chemotherapy is characterized in that:

takes damaged tumor cells, dead tumor cells and cell culture supernatant fluid treated by chemotherapy as a tumor antigen source, and prepares effective protein components in the tumor antigen into the nano vaccine with uniform particle size under the action of an organic solvent.

2. The method of claim 1, comprising the steps of:

s1: co-culturing tumor cells with 1-300 μ M chemotherapeutic drug for 1-4 days, and separating damaged tumor cells, dead tumor cells and cell culture supernatant;

s2: freezing and thawing the damaged tumor cells and the dead tumor cells in liquid nitrogen and 37 ℃ water bath for 2-10 times, centrifugally collecting cell lysate, and combining the cell lysate with the cell culture supernatant obtained in the step 1;

s3: concentrating the mixed solution obtained by the S2 by using an ultrafiltration cup with the molecular weight cutoff of 1-100kDa, washing by using deionized water without endotoxin, and quantifying by using a Bradford protein detection kit to obtain a protein solution;

s4: dropping organic solvent into the protein solution at the speed of 0.1-9 mL/min;

3. The method of claim 2, wherein:

in step S3, the concentration of the protein solution is 0.1-10mg/mL.

4. The production method according to claim 3, characterized in that:

in the step S4, the volume ratio of the organic solvent to the protein solution is 1-20:1.

5. the chemotherapy-induced tumor protein antigen nano-vaccine prepared according to the preparation method of any one of claims 1 to 4.

6. Use of the chemotherapy-induced tumor protein antigen nano-vaccine of claim 5, wherein:

the tumor protein antigen nano vaccine is used for preparing a therapeutic nano vaccine pharmaceutical preparation in the process of tumor treatment.

7. Use of the chemotherapy-induced tumor protein antigen nano-vaccine of claim 5, wherein:

the tumor protein antigen nano vaccine is used for preparing a therapeutic nano vaccine medicinal preparation and is used in combination with immunotherapy to exert a synergistic treatment effect.