T细胞受体CDR3δ移植性抗体（OT3）—免疫毒素的抗肿瘤研究及CDR3δ多肽的构象研究

发布时间：2018-06-26 05:12

本文选题：细胞 + 受体　；参考：《中国协和医科大学》2006年博士论文

【摘要】： 缺乏特异性肿瘤抗原是实体肿瘤抗体免疫治疗效果欠佳的重要原因;而γδT细胞能够经由其细胞受体(γδTCR)δ链的CDR3区(CDR3δ)广泛地识别、进而杀伤多种实体肿瘤细胞,比如卵巢癌。本试验将抗体分子重链的CDR3(CDR3H)用CDR3δ替换以获得γδT细胞的肿瘤识别能力,探索潜在的癌症治疗应用。本文首先用将CDR3H被替换为CDR3δ的IgG重链表达载体转染重链缺失型J558L骨髓瘤细胞,表达了抗体OT3Ab,其CDR3δ序列为本组报道的源自卵巢癌浸润Vδ2T细胞的OT3序列。经SDS-PAGE和Western Blotting验证后,流式细胞术分析显示OT3Ab能够和卵巢癌细胞系SKOV3特异结合(57.1%,CDR3未替换的对照抗体4.42%),核素~(99m)Tc标记OT3Ab后对接种SKOV3细胞的荷瘤裸鼠体内显像结果表明,OT3Ab能够特异聚集于肿瘤组织。随后本试验把OT3Ab和白喉毒素通过化学偶联的方法构建免疫毒素OT3AbDT,经免疫荧光显示其结合肿瘤细胞能力依然保存后,进行体内外抗肿瘤的试验治疗。蛋白合成抑制试验显示OT3AbDT对SKOV3细胞的IC_(50)介于1-10μg/ml。在SKOV3皮下荷瘤裸鼠模型中,瘤内注射OT3AbDT(13.8μg/次,每4天1次,共4次),荷瘤小鼠的肿瘤在16天后显著减小,和PBS差异显著。在更多种类的肿瘤皮下荷瘤模型中,包括人宫颈癌细胞系Hela、肝癌HepG2和直肠癌HR8348,尾静脉注射的OT3AbDT(13.8μg/次)也表现出不同程度的肿瘤体积增长被抑制的效果。上述结果表明CDR3δ移植性抗体为肿瘤的靶向免疫治疗提供了新的策略和工具。同时,本文也通过圆二色谱、分子动力学模拟对CDR3δ多肽的构象进行研究,为针对CDR3δ肽的研究提供必要的结构信息。
[Abstract]:The lack of specific tumor antigen is an important reason for the poor immunotherapy of solid tumor antibodies, and 纬未 T cells can be widely recognized by the CDR3 region (CDR3 未) of its cell receptor (纬未 TCR) 未 chain, which can kill many solid tumor cells, such as ovarian cancer. In this study, CDR3 (CDR3H) of antibody molecular heavy chain was replaced with CDR3 未 to obtain the tumor recognition ability of 纬未 T cells, and to explore the potential application of cancer therapy. In this paper, the IgG heavy chain expression vector of CDR3H was replaced with CDR3 未 and transfected into J558L myeloma cells, and the antibody OT3Abwas expressed. The CDR3 未 sequence was reported as OT3 sequence of ovarian cancer infiltrating V 未 2T cells. The results were confirmed by SDS-PAGE and Western blotting. Flow cytometry analysis showed that OT3Ab could specifically bind to ovarian cancer cell line SKOV3 (57.1% of the control antibody was not replaced by CDR3). In vivo imaging of nude mice inoculated with OT3Ab labeled with ~ (99m) Tc-labeled OT3Ab showed that OT3Ab could specifically concentrate in tumor tissue. Then the OT3Ab and diphtheria toxin were chemically coupled to construct the immunotoxin OT3AbDTT. After the immunofluorescence showed that OT3Ab was still preserved in combination with tumor cells, the anti-tumor therapy was carried out in vivo and in vitro. Protein synthesis inhibition test showed that the IC50 of OT3AbDT on SKOV3 cells ranged from 1 to 10 渭 g / ml. In SKOV3 subcutaneous tumor-bearing nude mice model, OT3AbDT (13.8 渭 g / time, once every 4 days, 4 times) was injected intratumoral, the tumor of tumor-bearing mice decreased significantly after 16 days, which was significantly different from that of PBS. In more subcutaneous tumor models, including human cervical cancer cell line Hela, liver cancer HepG2 and rectal cancer HR8348, OT3AbDT (13.8 渭 g / time) injected via caudal vein also showed the effect of inhibition of tumor volume growth in varying degrees. These results suggest that CDR3 未 transplantation antibody provides a new strategy and tool for tumor targeted immunotherapy. At the same time, the conformation of CDR3 未 peptide was studied by circular dichroism chromatography and molecular dynamics simulation, which provided necessary structural information for the study of CDR3 未 peptide.
【学位授予单位】：中国协和医科大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392

【相似文献】