DEC-205靶向的肝素酶CD4~+CD8~+T细胞表位肽纳米颗粒疫苗的抗肿瘤免疫效应研究
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摘要
目的探索肝素酶CD4~+CD8~+T细胞表位肽纳米颗粒疫苗诱导CTL抗肿瘤免疫反应的可行性,并研究其抗肿瘤免疫杀伤效应。方法根据文献报道方法制备肝素酶表位肽纳米颗粒疫苗;采用流式细胞术检测DEC-205靶向的肝素酶表位纳米颗粒疫苗与树突状细胞(DC)的结合力及入胞效率;采用标准4h~(51)Cr释放实验检测负载DEC-205靶向的肝素酶纳米颗粒疫苗诱导的效应细胞对胃癌及结肠癌细胞的杀伤效率;采用ELISA CD4~+CD8~+T细胞表位DEC-205靶向纳米颗粒疫苗,其颗粒直径为(208±15.3)nm,Zeta电位为(-28.8±2.5)mV;纳米颗粒对多肽的封包率为(22±3.6)%。纳米颗粒疫苗可与树突状细胞有效结合,并可以有效进入树突状细胞内,这种结合和内吞作用随着加入的纳米颗粒的浓度增加而增加。体外杀伤实验结果显示,在效靶比为80∶1时CD4~+CD8~+表位肽纳米颗粒疫苗诱导的效应细胞对肿瘤细胞的杀伤效率可达70%,与单用CD8~+表位肽相比具有统计学意义(P<0.01);T细胞增殖实验结果提示,CD4~+CD8~+表位肽组细胞的细胞增殖率显著高于单独使用CD8+表位肽组,细胞因子检测结果提示CD~4+CD8~+表位肽组细胞培养上清中IL-2、IL-12、IFN-γ浓度显著高于单独使用CD8~+表位肽组(P<0.01)。结论采用DEC-205靶向PLGA颗粒包裹CD4~+CD8~+表位肽能够更加有效递送抗原信息,能更加有效地诱导CTL反应杀伤肿瘤细胞,本研究为TAA为基础的肿瘤免疫治疗提供一种新的方式。
This study was designed to investigate the anti-tumor immune response elicited by DEC-205-targeting nano-particle packing CD4~+and CD8~+ epitopes derived from heparanase. Heparanase epitope peptide nanoparticle vaccine was prepared according to literature reports. Flow cytometry was used to detect the binding ability and internalization efficiency of DEC-205-targeting heparin epitope nanoparticle vaccine to dendritic cells(DC). The killing efficiency of effector cells induced by DEC-205-targeting heparinase nanoparticle vaccine against gastric cancer and colon cancer cells were detected by standard 4h~(51)Cr release assay; ELISA was employed to detect the cytokines secreted by effector cells. Data showed that the particle diameter is (208±15.3) nm, the zeta potential is(-28.8±2.5) mV, and the packing ratio of nanoparticles to polypeptide is(22±3.6)%.Nanoparticle vaccines can effectively bind to dendritic cells and can effectively enter dendritic cells, and this binding and endocytosis increased with the increase of nanoparticle concentration. The results of in vitro killing experiments showed that the killing efficiency of effector cells to tumor cells induced by CD4~+CD8~+epitope peptide nanoparticle vaccine was 70% at effector: target ratio of 80∶1, which was significant higher than that of CD8~+epitope peptide alone(P<0.01); the results of T cell proliferation assay showed that the cell proliferation rate of CD4~+CD8~+ epitope peptide cells was significantly higher than that of CD8~+ epitope peptide alone(P<0.01); cytokine detection results indicated that the levels of IL-2, IL-12 and IFN-γ in the cell culture supernatant of CD4~+CD8~+ epitope peptide group were significantly higher than those of CD8~+ epitope peptide alone(P<0.01). Taken together, DEC-205-targeting PLGA particles encapsulating CD4~+CD8~+ epitope peptides can more effectively deliver antigenic information and induce CTL responses to kill tumor cells, thus provides a new approach to TAA-based tumor immunotherapy.
This study was designed to investigate the anti-tumor immune response elicited by DEC-205-targeting nano-particle packing CD4~+and CD8~+ epitopes derived from heparanase. Heparanase epitope peptide nanoparticle vaccine was prepared according to literature reports. Flow cytometry was used to detect the binding ability and internalization efficiency of DEC-205-targeting heparin epitope nanoparticle vaccine to dendritic cells(DC). The killing efficiency of effector cells induced by DEC-205-targeting heparinase nanoparticle vaccine against gastric cancer and colon cancer cells were detected by standard 4h~(51)Cr release assay; ELISA was employed to detect the cytokines secreted by effector cells. Data showed that the particle diameter is (208±15.3) nm, the zeta potential is(-28.8±2.5) mV, and the packing ratio of nanoparticles to polypeptide is(22±3.6)%.Nanoparticle vaccines can effectively bind to dendritic cells and can effectively enter dendritic cells, and this binding and endocytosis increased with the increase of nanoparticle concentration. The results of in vitro killing experiments showed that the killing efficiency of effector cells to tumor cells induced by CD4~+CD8~+epitope peptide nanoparticle vaccine was 70% at effector: target ratio of 80∶1, which was significant higher than that of CD8~+epitope peptide alone(P<0.01); the results of T cell proliferation assay showed that the cell proliferation rate of CD4~+CD8~+ epitope peptide cells was significantly higher than that of CD8~+ epitope peptide alone(P<0.01); cytokine detection results indicated that the levels of IL-2, IL-12 and IFN-γ in the cell culture supernatant of CD4~+CD8~+ epitope peptide group were significantly higher than those of CD8~+ epitope peptide alone(P<0.01). Taken together, DEC-205-targeting PLGA particles encapsulating CD4~+CD8~+ epitope peptides can more effectively deliver antigenic information and induce CTL responses to kill tumor cells, thus provides a new approach to TAA-based tumor immunotherapy.
引文
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[12]Orbegoso C,Murali K,Banerjee S.The current status of immunotherapy for cervical cancer[J].Rep Pract Oncol Radiother,2018,23(6):580-588.
[13]Erhart F,Buchroithner J,Reitermaier R,et al.Immunological analysis of phase II glioblastoma dendritic cell vaccine(Audencel)trial:immune system characteristics influence outcome and Audencel upregulates Th1-related immunovariables[J].Acta Neuropathol Commun,2018,6(1):135.
[14]唐隽,郝飞.宫颈癌疫苗的现状和前景[J].免疫学杂志,2010,26(6):546-550.
[15]喻吉,林彩,李海波,等.人用疫苗佐剂及其免疫学机制研究现状[J].免疫学杂志,2018,34(9):811-817.
[16]Batty CJ,Tiet P,Bachelder EM,et al.Drug delivery for cancer immunotherapy and vaccines[J].Pharm Nanotechnol,2018,6(4):232-244.
[17]Moon JJ,Huang B,Irvine DJ.Engineering nano-and microparticles to tune immunity[J].Adv Mater,2012,24:3724-3746.
[18]Bozzacco L,Trumpfheller C,Siegal FP,et al.DEC-205receptor on dendritic cells mediates presentation of HIVgag protein to CD8+T cells in a spectrum of human MHC Ihaplotypes[J].Proc Natl Acad Sci U S A,2007,104:1289-1294.
[19]Allahyari M,Mohit E.Peptide/protein vaccine delivery system based on PLGA particles[J].Hum Vaccin Immunother,2016,12(3):806-828.
[20]Yang K,Liang Y,Sun Z,et al.T cell-derived lymphotoxin limits Th1 response during HSV-1 infection[J].Sci Rep,2018,8(1):17727.
[2]van Willigen WW,Bloemendal M,Gerritsen WR,et al.Dendritic cell cancer therapy:Vaccinating the right patient at the right time[J].Front Immunol,2018,9:2265.
[3]Zhang W,Lu X,Cui P,et al.Phase I/II clinical trial of a Wilms’tumor 1-targeted dendritic cell vaccination-based immunotherapy in patients with advanced cancer[J].Cancer Immunol Immunother,2019,68(1):121-130.
[4]Neek M,Kim TI,Wang SW.Protein-based nanoparticles in cancer vaccine development[J].Nanomed Nanotech Biol Med,2019,15(1):164-174.
[5]Kalos M,Levine BL,Porter DL,et al.T cells with chimeric antigen receptors have potent antitumor effects and can establish memory in patients with advanced leukemia[J].Sci Transl Med,2011,3(95):95ra73.
[6]Tang XD,Liang GP,Li C,et al.Cytotoxic T lymphocyte epitopes from human heparanase can elicit a potent antitumor immune response in mice[J].Cancer Immunol Immunother,2010,59(7):1041-1047.
[7]Tang XD,Wan Y,Chen L,et al.Immunogenic H-2Kbrestricted CTL epitopes from mouse heparanase elicit antitumor immune response in vivo[J].Cancer Res,2008,68(5):1529-1537.
[8]Cruz LJ,Tacken PJ,Rueda F,et al.Targeting nanoparticles to dendritic cells for immunotherapy[J].Methods Enzymol,2012,509:143-163.
[9]Cruz LJ,Tacken PJ,Bonetto F,et al.Multimodal imaging of nanovaccine carriers targeted to human dendritic cells[J].Mol Pharm,2011,8(2):520-531.
[10]Tang XD,Guo SL,Wang GZ,et al.In vitro and ex vivo evaluation of a multi-epitope heparinase vaccine for various malignancies[J].Cancer Sci,2014,105(1):9-17.
[11]Sarvizadeh M,Ghasemi F,Tavakoli F,et al.Vaccines for colorectal cancer:an update[J].J Cell Biochem,2018,[ahead of print].
[12]Orbegoso C,Murali K,Banerjee S.The current status of immunotherapy for cervical cancer[J].Rep Pract Oncol Radiother,2018,23(6):580-588.
[13]Erhart F,Buchroithner J,Reitermaier R,et al.Immunological analysis of phase II glioblastoma dendritic cell vaccine(Audencel)trial:immune system characteristics influence outcome and Audencel upregulates Th1-related immunovariables[J].Acta Neuropathol Commun,2018,6(1):135.
[14]唐隽,郝飞.宫颈癌疫苗的现状和前景[J].免疫学杂志,2010,26(6):546-550.
[15]喻吉,林彩,李海波,等.人用疫苗佐剂及其免疫学机制研究现状[J].免疫学杂志,2018,34(9):811-817.
[16]Batty CJ,Tiet P,Bachelder EM,et al.Drug delivery for cancer immunotherapy and vaccines[J].Pharm Nanotechnol,2018,6(4):232-244.
[17]Moon JJ,Huang B,Irvine DJ.Engineering nano-and microparticles to tune immunity[J].Adv Mater,2012,24:3724-3746.
[18]Bozzacco L,Trumpfheller C,Siegal FP,et al.DEC-205receptor on dendritic cells mediates presentation of HIVgag protein to CD8+T cells in a spectrum of human MHC Ihaplotypes[J].Proc Natl Acad Sci U S A,2007,104:1289-1294.
[19]Allahyari M,Mohit E.Peptide/protein vaccine delivery system based on PLGA particles[J].Hum Vaccin Immunother,2016,12(3):806-828.
[20]Yang K,Liang Y,Sun Z,et al.T cell-derived lymphotoxin limits Th1 response during HSV-1 infection[J].Sci Rep,2018,8(1):17727.