陈枢青教授与哈佛大学医学院周界文教授、吴皓教授合作在2019年第6期《细胞》(Cell)发表了题为“Higher-order clustering of the transmembrane anchor of DR5 drives signaling”的研究论文(https://doi.org/10.1016/j.cell.2019.02.0...陈枢青教授与哈佛大学医学院周界文教授、吴皓教授合作在2019年第6期《细胞》(Cell)发表了题为“Higher-order clustering of the transmembrane anchor of DR5 drives signaling”的研究论文(https://doi.org/10.1016/j.cell.2019.02.001)。该研究首次揭示了死亡受体5(death receptor 5,DR5)在激活后其跨膜区高度聚集,并直接激活下游的细胞凋亡通路。此外,该研究还提出DR5的胞外区在DR5激活前起到自我抑制作用,阻止DR5跨膜区的聚集倾向,从而防止受体由于跨膜区聚集而自动激活。该研究成功解释了部分二聚体或单体的抗体片段为何能够像三聚体的DR5肿瘤坏死因子相关凋亡诱导配体(TRAIL)那样激活DR5,而且其中某些DR5上的抗体表位与TRAIL在DR5上的结合位点完全不同。此外,肿瘤坏死因子受体(TNFR)家族包含了许多重要的免疫受体,比如TNFR2、OX40等,它们的跨膜区与DR5相似,均拥有GXXXG motif,可促进跨膜区聚集。因此,该研究提出了全新的TNFR家族受体的信号传递机制,阐明了单次跨膜跨膜区在信号传递过程中的重要作用,为DR5激动型靶向抗肿瘤药物的研发提供了思路,对于目前热门的免疫治疗具有积极的理论指导意义。展开更多
Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithioth...Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.展开更多
Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for develo...Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.展开更多
文摘陈枢青教授与哈佛大学医学院周界文教授、吴皓教授合作在2019年第6期《细胞》(Cell)发表了题为“Higher-order clustering of the transmembrane anchor of DR5 drives signaling”的研究论文(https://doi.org/10.1016/j.cell.2019.02.001)。该研究首次揭示了死亡受体5(death receptor 5,DR5)在激活后其跨膜区高度聚集,并直接激活下游的细胞凋亡通路。此外,该研究还提出DR5的胞外区在DR5激活前起到自我抑制作用,阻止DR5跨膜区的聚集倾向,从而防止受体由于跨膜区聚集而自动激活。该研究成功解释了部分二聚体或单体的抗体片段为何能够像三聚体的DR5肿瘤坏死因子相关凋亡诱导配体(TRAIL)那样激活DR5,而且其中某些DR5上的抗体表位与TRAIL在DR5上的结合位点完全不同。此外,肿瘤坏死因子受体(TNFR)家族包含了许多重要的免疫受体,比如TNFR2、OX40等,它们的跨膜区与DR5相似,均拥有GXXXG motif,可促进跨膜区聚集。因此,该研究提出了全新的TNFR家族受体的信号传递机制,阐明了单次跨膜跨膜区在信号传递过程中的重要作用,为DR5激动型靶向抗肿瘤药物的研发提供了思路,对于目前热门的免疫治疗具有积极的理论指导意义。
基金This work was supported by the grants from the National Key Research Project Funds，国家重点基础研究发展计划(973计划)
文摘Objective.Using monoclonal antibody (mAb) Fab′ fragment to develop mAb immunoconjugates for cancer. Methods.Fab′ fragment of mAb 3A5 was prepared by digestion of the antibody with pepsin and then reduced by dithiothreitol (DTT),while Fab′ fragment of mAb 3D6 was obtained by digestion of the antibody with ficin and subsequently reduced by β mercaptoethanol.The conjugation between Fab′ fragment and pingyangmycin (PYM),an antitumor antibiotic,was mediated by dextran T 40.Immunoreactivity of Fab′ PYM conjugates with cancer cells was determined by ELISA,and the cytotoxicity of those conjugates to cancer cells was determined by clonogenic assay.Antitumor effects of the Fab′ PYM conjugates were evaluated by subcutaneously transplanted tumors in mice. Results.The molecular weight of Fab′ fragment was approximately 53 kD,while the average molecular weight of Fab′ PYM conjugate was 170 kD.The Fab′ PYM conjugates showed immunoreactivity with antigen relevant cancer cells and selective cytotoxicity against target cells.Administered intravenously,Fab′ PYM conjugates were more effective against the growth of tumors in mice than free PYM and PYM conjugated with intact mAb. Conclusion.Fab′ PYM conjugate may be capable of targeting cancer cells and effectively inhibiting tumor growth,suggesting its therapeutic potential in cancer treatment.
基金This work was supported by the National Natural Sciences Founda- tion of China(NSFC, No. 39800057, No. 30200338) the National "863" High-tech Project Foundation (No. 102-10-01 -06) +1 种基金National Distinguished Youth Program of NSFC(No. 39525020) This wor
文摘Objective. To generate phage-displayed anti-idiotypic antibody single chain variable fragments (anti - Id ScFv) to MG7 monoclonal antibody (McAb) directed against gastric carcinoma so as to lay a foundation for developing anti-Id ScFv vaccine of the cancer.Methods. Balb/c mice were immunized i. p. with MG7 McAb conjugated with keyhole limpet hemocyanin (KLH), and mRNA was isolated from the spleens of the immunized mice. Heavy and light chain (VH and VL) genes of antibody were amplified separately and assembled into ScFv genes with a linker DNA by PCR. The ScFv genes were ligated into the phagemid vector pCANTAB5E and the ligated sample was transformed into competent E. coli TGI. The transformants were infected with M13K07 helper phage to yield recombinant phages displaying ScFv on the tips of M13 phage. After 4 rounds of panning with MG7, the MG7-positive clones were selected by ELISA from the enriched phages. The types of the anti-Id ScFv displayed on the selected phage clones were preliminarily identified by competition ELISA.Results. The VH, VL and ScFv DNAs were about 340 bp, 320 bp and 750 bp respectively. Twenty-four MG7-positive clones were selected from 60 enriched phage clones, among which 5 displayed β or γ type anti-Id ScFv.Conclusion. The anti-Id ScFv to MG7 McAb can be successfully selected by recombinant phage antibody technique, which paves a way for the study of prevention and cure of gastric carcinoma by using anti-Id ScFv.
