[1]张国秀,郑晨晨,赵智辉.特定激活性吞噬受体的配体与广谱肿瘤抗原的化学生物学桥接[J].南京师范大学学报(自然科学版),2020,43(02):92-99.[doi:10.3969/j.issn.1001-4616.2020.02.015]
 Zhang Guoxiu,Zheng Chenchen,Zhao Zhihui.Biochemical Bridging of Ligand for Specific Activating PhagocyticReceptor and Broad-Spectrum Tumor Antigens[J].Journal of Nanjing Normal University(Natural Science Edition),2020,43(02):92-99.[doi:10.3969/j.issn.1001-4616.2020.02.015]
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特定激活性吞噬受体的配体与广谱肿瘤抗原的化学生物学桥接()
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《南京师范大学学报》(自然科学版)[ISSN:1001-4616/CN:32-1239/N]

卷:
第43卷
期数:
2020年02期
页码:
92-99
栏目:
·生物学·
出版日期:
2020-05-30

文章信息/Info

Title:
Biochemical Bridging of Ligand for Specific Activating PhagocyticReceptor and Broad-Spectrum Tumor Antigens
文章编号:
1001-4616(2020)02-0092-08
作者:
张国秀郑晨晨赵智辉
南京师范大学生命科学学院,生物化学与生物制品研究所,江苏省分子与医学生物技术重点实验室,江苏 南京 210023
Author(s):
Zhang GuoxiuZheng ChenchenZhao Zhihui
School of Life Sciences,Nanjing Normal University,Institute of Biochemistry and Biological Products,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China
关键词:
代谢掺入生物正交反应激活性吞噬受体抗原递送肿瘤疫苗
Keywords:
metabolic incorporationbio-orthogonal reactionactivating phagocytic receptorantigen deliverytumor vaccine
分类号:
R730.3
DOI:
10.3969/j.issn.1001-4616.2020.02.015
文献标志码:
A
摘要:
目前肿瘤疫苗制备方法尚无法做到将广谱肿瘤抗原通过特定激活性吞噬受体途径递送给抗原呈递细胞,这可能是现有肿瘤疫苗临床疗效不佳的原因之一. 因此,本研究利用化学生物学方法,尝试将激活性吞噬受体的配体与广谱肿瘤抗原连接,为制备肿瘤疫苗提供新的方法. 首先,通过非天然糖代谢掺入对培养的小鼠4T1乳腺癌细胞和CT26.WT结直肠癌细胞抗原进行标记,使糖基化肿瘤抗原携带叠氮化的唾液酸,利用流式细胞术和免疫荧光技术确定代谢掺入的最优条件参数. 其次,利用生物正交反应使肿瘤抗原叠氮修饰位点再进一步共价缀合生物素,利用Western Blot检测蛋白的生物素化情况. 最后,利用抗原-抗体结合原理,将小鼠抗生物素单抗(IgG1亚型,其Fc结构域是IgG1FcR的配体)与生物素化肿瘤抗原交联形成免疫复合物,通过Western Blot判断最优交联参数. 实验结果表明,唾液酸前体物代谢掺入能高效地使肿瘤细胞发生叠氮化修饰,最佳浓度是2 mmol/L、最佳时间是24 h; 通过生物正交反应能使叠氮化肿瘤抗原进一步生物素化; 生物素化肿瘤抗原能高效地与抗生物素抗体形成复合物,二者交联的最佳比例(w/w)是20/1(4T1)或25/1(CT26.WT). 结果证明,本研究通过化学生物学手段实现了广谱肿瘤抗原与特定吞噬受体的配体的交联,在方法学上为研制新型肿瘤疫苗奠定了基础.
Abstract:
The current methods for preparing tumor vaccines has not been able to deliver broad-spectrum tumor antigens(TAgs)to antigen-presenting cells(APCs)through specific activating phagocytic receptor(SAPR)pathways,which may be one of the reasons for the poor clinical efficacy of existing tumor vaccines. Here,by using biochemical methods,it was tried to achieve the linkage of SAPR ligand with broad spectrum TAgs,attempting to provide a novel strategy for the preparation of tumor vaccines. Firstly,the antigens of 4T1 breast cancer cells or CT26.WT colorectal cancer cells were decorated by metabolic incorporation(MI)of non-natural carbohydrates,so that glycosylated tumor antigens could carry azide group on sialic acid residue,the optimal parameters of MI were determined by flow cytometry and immunofluorescent assay. Secondly,the azide modified sites of TAgs were further covalently conjugated with biotin through bio-orthogonal reaction,and the biotinylated tumor antigens(bio-TAgs)were detected by Western Blot. Finally,based on the principle of antigen-antibody binding,mouse anti-Biotin monoclonal antibody(anti-Biotin mAb,IgG1 subtype,whose Fc domain is a ligand of IgG1 FcR)was cross-linked with bio-TAgs to form immune complexes,and the optimal cross-linking parameters was determined by Western Blot. The results showed that TAgs were efficiently modified by azide through MI,the optimal cultural concentration of sialic acid precursor is 2 mmol/L,and the optimal time of MI is 24 h. The azide labeled TAgs can further become biotinylated via bio-orthogonal reaction,the bio-TAgs can efficiently cross-linked with the anti-Biotin mAb to form immune complexes,and the optimal ratio(w/w)of anti-Biotin mAb and bio-TAgs for cross-linkage are 20/1(4T1)or 25/1(CT26.WT). It is concluded that broad-spectrum tumor antigens can cross-link with SAPR ligand by biochemical methods,which lays a foundation for the development of new tumor vaccines.

参考文献/References:

[1] BRAY F,FERLAY J,SOERJOMATARAM I,et al. Global cancer statistics 2018:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J]. CA:a cancer journal for clinicians,2018,68(6):394-424.
[2]MARTIN L J,OCHOA DE O M,HIERRO C,et al. The expanding role of immunotherapy[J]. Cancer treatment reviews,2017,54:74-86.
[3]YE Z,QIAN Q,JIN H,et al. Cancer vaccine:learning lessons from immune checkpoint inhibitors[J]. Journal of cancer,2018,9(2):263-268.
[4]MELERO I,GAUDERNACK G,GERRITSEN W,et al. Therapeutic vaccines for cancer:an overview of clinical trials[J]. Nature reviews clinical oncology,2014,11(9):509-524.
[5]TAGLIAMONTE M,PETRIZZO A,TORNESELLO M L,et al. Antigen-specific vaccines for cancer treatment[J]. Human vaccines & immunotherapeutics,2014,10(11):3332-3346.
[6]ZIZZARI I G,VEGLIA F,TAURINO F,et al. HER2-based recombinant immunogen to target DCs through FcγRs for cancer immunotherapy[J]. Journal of molecular medicine,2011,89(12):1231-1240.
[7]OTT P A,HU Z,KESKIN D B,et al. An immunogenic personal neoantigen vaccine for patients with melanoma[J]. Nature,2017,547(7662):217-221.
[8]SABADO R L,BALAN S,BHARDWAJ N. Dendritic cell-based immunotherapy[J]. Cell research,2017,27(1):74-95.
[9]SAKAI K,SHIMODAIRA S,MAEJIMA S,et al. Dendritic cell-based immunotherapy targeting Wilms’ tumor 1 in patients with recurrent malignant glioma[J]. Journal of neurosurg,2015,123(4):989-997.
[10]RONG J,HAN J,DONG L,et al. Glycan imaging in intact rat hearts and glycoproteomic analysis reveal the upregulation of sialylation during cardiac hypertrophy[J]. Journal of the American chemical society,2014,136(50):17468-17476.
[11]ROUHANIFARD SH,NORDSTROM L U,ZHENG T,et al. Chemical probing of glycans in cells and organisms[J]. Chemical society reviews,2013,42(10):4284-4296.
[12]MILANI A,SANGIOLO D,AGLIETTA M,et al. Recent advances in the development of breast cancer vaccines[J]. Breast cancer,2014,6:159-168.
[13]NAGORSEN D,THIEL E. Clinical and immunologic responses to active specific cancer vaccines in human colorectal cancer[J]. Clinical cancer research:an official journal of the American association for cancer research,2006,12(10):3064-3069.
[14]RAO B,HAN M,WANG L,et al. Clinical outcomes of active specific immunotherapy in advanced colorectal cancer and suspected minimal residual colorectal cancer:a meta-analysis and system review[J]. Journal of translational medicine,2011,9:17-27.
[15]PERDICCHIO M,ILARREGUI J M,VERSTEGE M I,et al. Sialic acid-modified antigens impose tolerance via inhibition of T-cell proliferation and de novo induction of regulatory T cells[J]. Proceedings of the national academy of sciences of the United States of America,2016,113(12):3329-3334.
[16]RIAD A,CHARLES A D,LAUREN A,et al. Activating and inhibitory Fcgamma receptors in immunotherapy:being the actor or being the target[J]. Expert review of clinical immunology,2009,5(6):735.
[17]SONDERMANN P. The FcγR/IgG interaction as target for the treatment of autoimmune diseases[J]. Journal of clinical immunology,2016,36(1):95-99.
[18]HU Z,OTT P A,WU C J. Towards personalized,tumour-specific,therapeutic vaccines for cancer[J]. Nature reviews immunology,2018,18(3):168-182.

备注/Memo

备注/Memo:
收稿日期:2019-12-15.
基金项目:国家自然科学基金重大研究计划集成项目子课题(2013104GZ90073).
通讯作者:赵智辉,博士,教授,研究方向:肿瘤疫苗治疗. E-mail:zhaozhihui_1964@aliyun.com
更新日期/Last Update: 2020-05-15