Background:Chimeric antigen receptor T(CAR-T)cell therapies have demonstrated significant clinical efficacy in hematological malignancies.However,their application to solid tumors remains substantially limited by mult...Background:Chimeric antigen receptor T(CAR-T)cell therapies have demonstrated significant clinical efficacy in hematological malignancies.However,their application to solid tumors remains substantially limited by multiple challenges,including the risk of off-target effects.Hence,optimizing CAR-T cells for stronger antigen binding is essential.Methods:In this study,we employed a classical anti-human endothelial growth factor receptor 2(HER2)single-chain variable fragment(scFv)derived from trastuzumab,alongside an anti-HER2-13 scFv identified from a combinatorial cellular CAR library,for the construction of a third-generation CAR-T cell.Meanwhile,the phenotypes and both in vitro and in vivo functions of CAR-T cells transduced with the two scFvs via PiggyBac transposon-mediated gene transfer were compared.Results:The optimal ratio between the PiggyBac HER2-CAR-puro transposon and the Super PiggyBac transposase plasmid differed during the construction of the two HER2-targeted CAR-T cell types.The expansion abilities,CD3^(+)CAR^(+)population,CD4^(+)CAR^(+)/CD8^(+)CAR^(+)proportions,and memory and exhaustion markers between the two CAR-T groups were similar after using the optimized proportion of plasmid.Both CAR-T cell types exhibited significant antitumor activity,with the anti-HER2-13 CAR-T cells demonstrating superior target specificity.Therapeutic effects were observed with both CAR-T cells and trastuzumab in theMDA-MB-231HER2+breast tumor xenograft model,with anti-HER2-13 CAR-T cells demonstrating slightly enhanced efficacy and no evident offtarget toxicity.Conclusion:These results highlight the potential of anti-HER2-13 CAR-T cells to serve as a safer and more efficacious alternative in HER2-targeted therapy.展开更多
将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产...将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产生荧光蚕的比例分别为0.17%,0.15%。将次代荧光蚕与正常蚕交配后代(G1)的荧光蚕个体再相互杂交,连续进行多代选育,获得了稳定遗传的转hGM-CSF基因家蚕品系。展开更多
基金supported by the Yunnan Fundamental Research Projects,China(Grant No.202201AS070068)Central Funds Guiding the Local Science and Technology Development,China(202207AB110017)+1 种基金The Science and Technology Fund of Kunming City,China(No.2019-1-N-25318000002027)The Scientific and Technological Innovation Team in Kunming Medical University,China(CXTD202215).
文摘Background:Chimeric antigen receptor T(CAR-T)cell therapies have demonstrated significant clinical efficacy in hematological malignancies.However,their application to solid tumors remains substantially limited by multiple challenges,including the risk of off-target effects.Hence,optimizing CAR-T cells for stronger antigen binding is essential.Methods:In this study,we employed a classical anti-human endothelial growth factor receptor 2(HER2)single-chain variable fragment(scFv)derived from trastuzumab,alongside an anti-HER2-13 scFv identified from a combinatorial cellular CAR library,for the construction of a third-generation CAR-T cell.Meanwhile,the phenotypes and both in vitro and in vivo functions of CAR-T cells transduced with the two scFvs via PiggyBac transposon-mediated gene transfer were compared.Results:The optimal ratio between the PiggyBac HER2-CAR-puro transposon and the Super PiggyBac transposase plasmid differed during the construction of the two HER2-targeted CAR-T cell types.The expansion abilities,CD3^(+)CAR^(+)population,CD4^(+)CAR^(+)/CD8^(+)CAR^(+)proportions,and memory and exhaustion markers between the two CAR-T groups were similar after using the optimized proportion of plasmid.Both CAR-T cell types exhibited significant antitumor activity,with the anti-HER2-13 CAR-T cells demonstrating superior target specificity.Therapeutic effects were observed with both CAR-T cells and trastuzumab in theMDA-MB-231HER2+breast tumor xenograft model,with anti-HER2-13 CAR-T cells demonstrating slightly enhanced efficacy and no evident offtarget toxicity.Conclusion:These results highlight the potential of anti-HER2-13 CAR-T cells to serve as a safer and more efficacious alternative in HER2-targeted therapy.
文摘将由家蚕核型多角体病毒IE-1基因启动子控制下的hGM-CSF基因克隆到p igA3GFP载体中,构建了家蚕转基因载体p igA3GFP[IE-GMCSF],利用压力渗透法和精子介导法将其与辅助质粒helper p igA3一起导入家蚕蚕卵,获得产生绿色荧光的家蚕,次代产生荧光蚕的比例分别为0.17%,0.15%。将次代荧光蚕与正常蚕交配后代(G1)的荧光蚕个体再相互杂交,连续进行多代选育,获得了稳定遗传的转hGM-CSF基因家蚕品系。
