Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs
阅读说明：本技术 一种癌干细胞样耐药细胞来源泛素化蛋白及其在制备抗癌药物中的应用 (Cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparation of anti-cancer drugs ) 是由 王立新 黄芳 于 2019-10-12 设计创作，主要内容包括：本发明公开了一种癌干细胞样耐药细胞来源泛素化蛋白及其在制备抗癌药物中的应用,其主要由以下方法制备所得：(1)利用化疗药诱导癌细胞耐药,得到富集了癌干细胞的癌干细胞样耐药细胞株；(2)将泛素化蛋白募集蛋白偶联至佐剂,得偶联佐剂；(3)培养上述癌干细胞样耐药细胞株,抑制其泛素化蛋白降解,然后利用步骤(2)所得的偶联佐剂募集泛素化蛋白,即得所述泛素化蛋白。本发明在原有基础上改变了泛素化蛋白来源,制备获得了具有癌干细胞样耐药肿瘤细胞抗原特异性的泛素化蛋白,诱导了具有癌干细胞样耐药肿瘤细胞特异性的免疫应答,并且联合STING激动剂,大大提高疫苗的治疗肿瘤的效果。(The invention discloses a cancer stem cell-like drug-resistant cell-derived ubiquitination protein and application thereof in preparation of anti-cancer drugs, and the cancer stem cell-like drug-resistant cell-derived ubiquitination protein is mainly prepared by the following method: (1) inducing cancer cell drug resistance by using chemotherapeutic drugs to obtain a cancer stem cell-like drug-resistant cell strain enriched with cancer stem cells; (2) coupling the ubiquitinated protein recruiting protein to an adjuvant to obtain a coupled adjuvant; (3) culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitinated protein, and then recruiting the ubiquitinated protein by using the coupling adjuvant obtained in the step (2) to obtain the ubiquitinated protein. The invention changes the source of ubiquitinated protein on the original basis, prepares and obtains ubiquitinated protein with the specificity of cancer stem cell-like drug-resistant tumor cell antigen, induces the immune response with the specificity of cancer stem cell-like drug-resistant tumor cell, and greatly improves the tumor treatment effect of the vaccine by combining with STING agonist.)
1. A cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is characterized in that the drug-resistant cell-derived ubiquitinated protein is mainly prepared by the following method:
(1) establishment of cancer-resistant cells: inducing cancer cell drug resistance by using chemotherapeutic drugs to obtain a cancer stem cell-like drug-resistant cell strain enriched with cancer stem cells;
(2) modification of the adjuvant: coupling the ubiquitinated protein recruiting protein to an adjuvant to obtain a coupled adjuvant;
(3) recruiting ubiquitinated proteins: culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitination protein, and then collecting ubiquitination protein by using the coupling adjuvant obtained in the step (2), so as to obtain ubiquitination protein derived from the cancer stem cell-like drug-resistant cell.
2. The cancer stem cell-like drug-resistant cell-derived ubiquitinated protein of claim 1, wherein in step (1), the cancer cell is a breast cancer cell and the chemotherapeutic is selected from the group consisting of alkylating agents, antimetabolite drugs, antibiotics, alkaloids or taxanes, preferably the antibiotic epirubicin.
3. The cancer stem cell-like drug-resistant cell-derived ubiquitinated protein of claim 1, wherein in step (1), the cancer cell-like drug resistance is induced by a combination of low-concentration gradient increment and a high-dose intermittent impact method, preferably by the following method:
culturing cancer cells, changing into a culture medium containing chemotherapeutic drugs when the cells grow to 55-85%, gradually increasing the drug-containing concentration of the culture medium from (0.01-5) mu g/ml to (100) -500 mu g/ml, culturing, changing the culture medium once every 1-3 days, and carrying out passage.
4. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 1, wherein in step (2), the ubiquitinated protein recruiting protein is selected from a ubiquitin antibody or a ubiquitin binding protein, preferably a ubiquitin binding protein, further preferably a Vx3 protein;
the adjuvant is selected from a nano calcium phosphate adjuvant, a nano ferroferric oxide adjuvant, a manganese adjuvant or nano aluminum, and the nano aluminum is preferred.
5. The cancer stem cell-like resistant cell-derived ubiquitinated protein of claim 1, wherein in the step (3), the method for inhibiting degradation of ubiquitinated protein comprises: culturing the cells to grow to 75-85%, adding a proteasome inhibitor and ammonium chloride in the amount of (200-500) nmol/L and (50-100) mmol/L respectively, and interfering the cells for 100-100 hours to inhibit the degradation of ubiquitinated protein.
6. Use of the cancer stem cell-like resistant cell-derived ubiquitinated protein of any one of claims 1 to 5 for the preparation of an anticancer drug.
7. The use according to claim 6, wherein the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is used for preparing a drug for treating drug-resistant metastatic cancer.
8. Use of the cancer stem cell-like resistant cell-derived ubiquitinated protein of any one of claims 1 to 5 in combination with Sting agonists for the preparation of an anti-cancer medicament.
9. The use of claim 8, wherein the cancer stem cell-like drug-resistant tumor cell-derived ubiquitinated protein is used in combination with Sting agonists for preparing a drug for treating drug-resistant metastatic cancer.
10. The use according to claim 8, wherein the Sting agonist is selected from DMXAA or 2'3' -c-di-Amp.
Technical Field
The invention relates to a cancer stem cell-like drug-resistant cell-derived ubiquitination protein and application thereof in preparation of anti-cancer drugs, belonging to the technical field of tumor vaccines.
Background
With the continuous development of oncology, immunology, molecular biology and other disciplines, tumor immunotherapy is becoming an emerging treatment method beyond traditional treatments such as surgery, chemotherapy, radiotherapy and the like. Tumor immunotherapy kills tumors, inhibits tumor growth and metastasis by stimulating and enhancing the body's own anti-tumor immune response. In clinical studies, immunotherapy has achieved dramatic results in melanoma, lung cancer, ovarian cancer, breast cancer, etc., and some immunotherapies have received FDA approval in the united states. Taking breast cancer as an example, breast cancer still is a common cancer of women at present, and is the second leading cause of death of women, and the incidence rate is also increased year by year. The current conventional treatment for breast cancer includes conventional cancer treatment methods using surgery, radiation therapy and chemotherapy, and also includes targeted therapy and hormone therapy. Clinical data show that breast cancer has high heterogeneity, high metastasis, easy recurrence and poor prognosis, most tumor patients are not sensitive to chemotherapy, radiotherapy and the like, long-term chemotherapy and targeted therapy can also cause drug resistance of breast cancer patients, and particularly for patients who appear or develop into stage IV diseases, the 5-year survival rate is still poor. Therefore, new means and treatment strategies are urgently needed for clinical treatment of breast cancer, and the emergence of immunotherapy in recent years is expected to become a new opportunity for treating breast cancer. Immunotherapy including tumor vaccines has the advantages of strong targeting property, small toxic and side effects and the like, and clinically tests with a plurality of achievements are achieved, and especially therapeutic tumor vaccines are more and more concerned by people.
The important factor for the tumor vaccine to play a role is the tumor-associated antigens (TAAs), the tumor vaccine is mainly formed by cross presenting the TAAs by professional antigen presenting cells (pAPC), inducing initial T cell response, recognizing the TAAs by activated T cells and specifically killing target cells. The applicant finds that autophagosome DRibble rich in ubiquitination protein is an effective antigen carrier, tumor antigen carried by the DRibble can be taken up by DC, and specific CD8 is induced by a cross-presentation mode+T cell response. Further research finds that ubiquitinated protein Ub is a main component of DRibble playing an anti-tumor role, and applicant can directly enrich ubiquitinated protein in tumor cell lysate rich in ubiquitinated protein on various tumor models through Vx3 fusion protein (His-Vx3-eGFP) with three ubiquitin molecule binding motifs (UIM) in the early periodHas antitumor effect. In order to further optimize the ubiquitinated protein vaccine, the applicant covalently couples His-Vx3-eGFP protein to nano aluminum, and by utilizing the physical characteristics of the nano aluminum, the ubiquitinated protein can be quickly, simply and efficiently recruited by a one-step centrifugation method, and meanwhile, the nano aluminum is used as an immune adjuvant, so that the immune effect of the ubiquitinated protein is further improved, the ubiquitinated protein-nano aluminum adjuvant composite vaccine is successfully prepared, and the anti-tumor effect can be effectively exerted on a tumor-bearing mouse model.
The breast cancer patients are easy to have metastasis and generate tumor drug resistance, cancer stem-like breast cancer tumor cells are enriched in the drug-resistant tumor cells, and the cancer stem cells are a small part of cell groups which exist in tumor tissues and have self-renewal, proliferation and differentiation functions and are considered as key factors and even sources of tumor recurrence, metastasis and drug resistance. The development of a new treatment strategy taking cancer stem cells as targets, the elimination of the cancer stem cells which are mainly targeted while paying attention to the overall reduction of the tumor volume is expected to overcome the occurrence of tumor recurrence, metastasis and drug tolerance, and the new concept in the field of tumor treatment is formed.
The existing conventional therapeutic tumor vaccine is usually aimed at primary drug-intolerant non-metastatic breast cancer, and can not play an effective role in some breast cancer patients who are in late stage or have metastasis or drug resistance.
Disclosure of Invention
The purpose of the invention is as follows: in order to overcome the defects of the prior art, the invention aims to provide a cancer stem cell-like drug-resistant cell-derived ubiquitinated protein and application thereof in preparing an anti-cancer drug, in particular application of the ubiquitinated protein in combination with Sting agonist DMXAA in treating metastatic cancer.
The technical scheme is as follows: in order to achieve the purpose, the invention adopts the following technical scheme:
a cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is mainly prepared by the following steps:
(1) establishment of cancer-resistant cells: inducing cancer cell drug resistance by using chemotherapeutic drugs to obtain a cancer stem cell-like drug-resistant cell strain enriched with cancer stem cells;
(2) modification of the adjuvant: coupling the ubiquitinated protein recruiting protein to an adjuvant to obtain a coupled adjuvant;
(3) recruiting ubiquitinated proteins: culturing the cancer stem cell-like drug-resistant cell strain, inhibiting degradation of ubiquitinated protein, and then collecting ubiquitinated protein by using the coupling adjuvant obtained in the step (2), so as to obtain the ubiquitinated protein derived from the cancer stem cell-like drug-resistant cell.
In step (1), the cancer cells are preferably breast cancer cells, and the chemotherapeutic drug may be selected from clinical commonly used drugs for breast cancer patients, including alkylating agents (such as cyclophosphamide, melphalan, etc.), antimetabolic drugs (such as fluorouracil, methotrexate, etc.), antibiotics (such as adriamycin, epirubicin, mitomycin C, etc.), alkaloids (such as vincristine, colchicine, etc.), and taxols (such as docetaxel, etc.), preferably the antibiotics epirubicin. Other similar cancer cells are also suitable, for example, the cancer cells can also be selected from lung cancer cells, and the chemotherapy drug cisplatin is used for inducing the drug resistance of the lung cancer cells, and the like.
In the step (1), the drug resistance of the cancer cells is induced by combining a low concentration gradient increment method with a high-dose intermittent impact method, and the method is preferably as follows:
culturing cancer cells, changing into a culture medium containing chemotherapeutic drugs when the cells grow to 55-85%, gradually increasing the drug-containing concentration of the culture medium from (0.01-5) mu g/ml to (100) -500 mu g/ml, culturing, changing the culture medium once every 1-3 days, and carrying out passage.
In step (2), the ubiquitinated protein recruiting protein may be selected from ubiquitin antibodies (e.g., ubiquitin Antibody, K11-linkage Specific Polyubiquitin Antibody, K48-linkage Specific Polyubiquitin Antibody, K63-linkage Specific Polyubiquitin Antibody, etc.) or ubiquitin binding proteins, preferably ubiquitin binding proteins such as Vx3 protein, etc., more preferably Vx3 protein.
For preparation and purification of Vx3 protein, tag protein such as His protein, MyC protein, GST protein or HA protein can be selected for labeling, preferably His protein.
The adjuvant is selected from a nano calcium phosphate adjuvant, a nano ferroferric oxide adjuvant, a manganese adjuvant or nano aluminum, and the nano aluminum is preferred.
Vx3 protein can be adsorbed or covalently coupled to nano-aluminum adjuvant, preferably by covalent coupling method with coupling agent. More preferably, the coupling agent is selected from triethoxysilane, which is linked to the nano-aluminum adjuvant through an aldehyde group and then reacts with His-Vx3 protein to generate a stable carbon-nitrogen double bond through an amino group and a carboxyl group.
The His-Vx3 protein can be prepared by the following method:
and (3) recovering the Escherichia coli transformed with the His-Vx3 expression plasmid, inducing the expression protein of the Escherichia coli, and extracting the His-Vx3 protein by adopting a nickel ion chromatography.
The His-Vx3 protein is a Vx3(A7) fusion protein with a ubiquitin binding domain, the Vx3(A7) fusion protein is an artificial construct which is connected in series with three UIMs (ubiquitin-interacting moieties) derived from Vps27(a yeast homology of HRS, Hepatocyte growth factor-regulated tyrosine kinase substrate), and the protein is a protein which is reported in recent years and is artificially constructed and connected in series with the ubiquitin binding domain and has high affinity to ubiquitinated protein.
In the step (3), the method for inhibiting the degradation of ubiquitinated protein is preferably: the cultured cancer stem cell-like drug-resistant cells grow to 75-85%, a proteasome inhibitor (such as PSI, lactacystin, MG132 or bortezomib, preferably bortezomib) and ammonium chloride are added, the addition amount is respectively (200-.
And (3) after inhibiting degradation of ubiquitinated protein in the drug-resistant cells, treating the cells by using a cell lysate, centrifuging to obtain a supernatant, incubating the supernatant with the coupled nano aluminum particles to collect ubiquitinated protein, and centrifuging at a low temperature (2-15 ℃) to obtain a precipitate, namely the ubiquitinated protein derived from the cancer stem cell-like drug-resistant cells. The co-incubation is preferably performed overnight at 4 ℃.
The invention also provides application of the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein in preparation of anti-cancer drugs. Preferably, when the ubiquitinated protein source is a breast cancer cell, it can be used to treat breast cancer.
Preferably, the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is used for preparing a drug for treating drug-resistant metastatic cancer, and further preferably, when the ubiquitinated protein is derived from breast cancer cells, the ubiquitinated protein can be used for treating drug-resistant metastatic breast cancer.
The invention finally provides application of the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein combined Sting agonist in preparation of anti-cancer drugs. Preferably, when the ubiquitinated protein source is breast cancer cells, the combination of the ubiquitinated protein and the Sting agonist can be used for treating breast cancer, and the effect is more remarkable.
Preferably, the cancer stem cell-like drug-resistant cell-derived ubiquitinated protein is combined with Sting agonist to be applied to preparation of drugs for treating drug-resistant metastatic cancer. Preferably, when the ubiquitinated protein source is a breast cancer cell, the combination of the ubiquitinated protein and a Sting agonist can be used for treating drug-resistant metastatic breast cancer, and the effect is more remarkable.
The Sting agonist is selected from DMXAA or 2'3' -c-di-Amp, etc., or 2'3' -cGAMP, 3'3' -cGAMP, c-di-AMP, etc., which have similar structure to 2'3' -c-di-Amp, etc., and preferably DMXAA or 2'3' -c-di-Amp.
Aiming at drug-resistant and multiple metastatic cancer patients generated by clinical chemotherapy, particularly breast cancer patients, the invention prepares a novel vaccine of drug-resistant cell-derived ubiquitinated protein in a cancer stem cell shape by utilizing a highly metastatic mouse 4T1 model simulating human IV-stage breast cancer, solves the problems of drug resistance and high metastasis of breast cancer tumor, and explores the anti-tumor effect and mechanism of the novel vaccine. The method comprises the steps of inducing cancer stem cell-like epirubicin-resistant cells by an in vitro low-concentration gradient increment combined large-dose intermittent impact method, further identifying drug resistance and cancer stem cell characteristics in vivo and in vitro, successfully inducing the cancer stem cell-like epirubicin-resistant cells 4T1/EPB, and establishing a breast cancer drug-resistant metastatic tumor-bearing mouse model through the 4T1/EPB cells.
In addition, the invention connects the ubiquitinated protein of the drug-resistant tumor cell source of the cancer stem cell sample with the nano-aluminum to prepare a novel vaccine on the basis of the original vaccine, combines with Sting agonist DMXAA and the like, explores a new immunotherapy strategy, researches and observes the treatment effect on a drug-resistant high-metastatic breast cancer 4T1/EPB mouse model, and provides an experimental basis for the clinical research of the treatment of multiple chemotherapy drug-resistant and metastatic cancer patients.
Compared with the existing research, the research of the invention has the following advantages:
1. the vaccine alpha-Al prepared by the invention2O3The alpha-Al of the previous generation nano aluminum vaccine is greatly improved by the-His-Vx 3-Ub (4T1/EPB)2O3The anti-tumor effect of His-Vx3-Ub (4T1/WT) on 4T1/WT breast cancer tumor-bearing mice obviously prolongs the survival time of the mice;
2. the vaccine of the invention alpha-Al2O3His-Vx3-Ub (4T1/EPB) in 4T1/EPB drug-resistant highly metastatic breast cancer tumor-bearing mice can effectively inhibit tumor growth and tumor lung metastasis and remarkably prolong the survival time of the mice;
3. the vaccine of the invention alpha-Al2O3His-Vx3-Ub (4T1/EPB) combined with Sting agonist DMXAA or 2'3' -c-di-Amp can cause the regression of most of mouse tumors in 4T1/EPB drug-resistant highly metastatic breast cancer tumor-bearing mice, and tumor-regressed tumor-bearing mice have immunological memory and can resist the re-attack of 4T1/EPB tumors.
The technical effects are as follows: compared with the prior art, the method changes the source of the ubiquitinated protein on the basis of the prior art, prepares the ubiquitinated protein with the specificity of the cancer stem cell-like drug-resistant tumor cell antigen, induces the immune response with the specificity of the cancer stem cell-like drug-resistant tumor cell, and combines the STING agonist, thereby greatly improving the treatment effect of the vaccine, and particularly having more remarkable treatment effect on the drug-resistant metastatic cancer.
Drawings
FIG. 1: induction and drug resistance identification of mouse breast cancer epirubicin
FIG. 2: identification of mouse breast cancer epirubicin resistant cell 4T1/EPB cancer stem cell: A.4T1/EPB cancer stem cell surface marker CD44+CD24-/lowDetection of the subpopulation; detecting a 4T1/EPB cancer stem cell marker acetaldehyde dehydrogenase 1(ALDH 1); C.4T1/EPB migration ability detection; D.4T1/EPB invasion capacity detection; E.4T1/EPB tumorigenicity detection.
FIG. 3: alpha-Al2O3His-Vx 3-recruitment of ubiquitinated proteins from 4T1/WT and 4T1/EPB cells, respectively; A. detecting the loading amount of the sample protein; detection of the expression of ub.
FIG. 4: alpha-Al2O3Detection of the anti-tumor effect of the His-Vx3-Ub (4T1/EPB) vaccine; A. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3His-Vx3-Ub (4T1/EPB) vaccine treated the tumor growth curve of mice in a 4T1/WT tumor-bearing mouse model; B. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3-the survival time of His-Vx3-Ub (4T1/EPB) vaccine treated mice in a 4T1/WT tumor-bearing mouse model; C. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3His-Vx3-Ub (4T1/EPB) vaccine for treatment of metastasis in mice in a 4T1/WT tumor-bearing mouse model; D. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3His-Vx3-Ub (4T1/EPB) vaccine treated the tumor growth curve of mice in a 4T1/EPB tumor-bearing mouse model; E. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3-the survival time of His-Vx3-Ub (4T1/EPB) vaccine treated mice in a 4T1/WT tumor-bearing mouse model; F. alpha-Al2O3His-Vx3-Ub (4T1/WT) and alpha-Al2O3His-Vx3-Ub (4T1/EPB) vaccine treated mice for metastasis in the 4T1/WT tumor-bearing mouse model.
FIG. 5: observation of alpha-Al2O3His-Vx3-Ub (4T1/EPB) and alpha-Al2O3Detection of the immune response induced by His-Vx3-Ub (4T1/WT) and its specificity: A. CD3 in spleen cells of mice after different immunizations+CD8+IFN-γ+The proportion of T cells of (a); B. is not limited toCD3 in spleen cells of mice after isoimmunization+CD8+IFN-γ+(iii) statistics of T cell ratios of (a); C. and (3) detecting the IFN-gamma level in spleen cells of mice after different immunizations.
FIG. 6: alpha-Al2O3Detection of the Effect of His-Vx3-Ub (4T1/EPB) vaccine Mixed Sting agonists on BMDC: A. alpha-Al2O3Detection of BMDC secreting type I interferon IFN- β by His-Vx3-Ub (4T1/EPB) mixed Sting agonist 2'3' -c-di-Amp; BETA, ALPHA-Al2O3Detection of BMDC secreting type I interferon IFN- β by His-Vx3-Ub (4T1/EPB) mixed Sting agonist DMXAA.
FIG. 7: alpha-Al2O3Detection of the anti-tumor effect of His-Vx3-Ub (4T1/EPB) vaccine in combination with Sting agonist DMXAA; A. alpha-Al2O3-tumor growth curve of His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA for treatment of 4T1/EPB tumor-bearing mice; B. alpha-Al2O3-the survival time of 4T1/EPB tumor-bearing mice treated with His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA; C. alpha-Al2O3-transfer profile of His-Vx3-Ub (4T1/WT) vaccine in combination with Sting agonist DMXAA for treatment of 4T1/EPB tumor-bearing mice; D. alpha-Al2O3Detection of immunological memory induced by His-Vx3-Ub (4T1/EPB) vaccine in combination with Sting agonist DMXAA.
Detailed Description
The present invention will be further illustrated with reference to the following specific examples.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：猪口蹄疫病毒A型抗体酶联免疫检测试剂盒