图1 CAR载体示意图 4-1BB和CD3ζ为CAR分子细胞内共刺激信号,scFv 为靶向特定抗原的单链抗体片段,后4种载体的CAR分子通过T2A系统与绿色荧光蛋白共表达。（Dupixent-HL-GFP中scFv部分重链排在轻链前,Dupixent-LH-GFP中scFv部分轻链排在重链前）

Fig. 1 Schematic diagram of CAR（chimeric antigen rec-eptor）vectors 4-1BB and CD3ζ are intracellular costimulatory signals of CAR molecules,and scFv is single-chain antibody fragment targeting specific antigens. The CAR molecules in the latest 4 vectors co-express with green fluorescent protein by the T2A system.（The scFv in Dupixent-HL-GFP is arranged in an order of the heavy chain followed by the light chain,and the scFv in Dupixent-LH-GFP is arranged in an order of the light chain followed by the heavy chain）

图2 CAR载体酶切图 上方条带为载体骨架,下方条带为图1所示的CAR分子部分

Fig. 2 Map of restriction enzyme digestion of CAR vectors The upper band is the vector backbone,and the lower band is the CAR DNA schemed in Fig. 1 M:Marker. 1:Meso-CAR. 2:Met-CAR. 3:CAR1-2-GFP. 4:CAR2-4-GFP. 5:Dupixent-HL-GFP. 6:Dupixent-LH-GFP

图3 CAR分子scFv段测序图谱（部分）

Fig. 3 Sequencing profiles of scFv segments in CAR molecules（partial）

图4 流式细胞仪检测CAR表达结果 在免疫球蛋白G抗体染色中Met-CAR,Dupixent-HL-GFP和Dupixent-HL-GFP细胞使用的一抗为Biotin-SP Rabbit Anti-Human IgG;Meso-CAR,CAR1-2-GFP和CAR2-4-GFP使用的一抗为Biotin-SP Goat Anti-Mouse IgG。在蛋白质 L染色中,6种细胞使用的一抗为Biotin-protein L。这两种染色方法使用的二抗均为PE-Streptavidin。前两行数据中,空心峰为同时使用一抗和荧光二抗的实验组,实心峰为仅加荧光二抗的对照组;第三行数据中,实心峰为Jurkat细胞,空心峰为GFP与CAR共表达的4种细胞。纵坐标为细胞数量,横坐标为荧光强度,数据显示检测到的CAR分子阳性率

Fig. 4 Results of CAR expression detected by flow cytometry The primary antibody used for Met-CAR,Dupixent-HL-GFP and Dupixent-HL-GFP cells in the immunoglobulin G antibody staining assay is Biotin-SP Rabbit anti-Human IgG,while that for Meso-CAR,CAR1-2-GFP and CAR2-4-GFP is Biotin-SP Goat Anti-Mouse IgG. In protein L staining assay,the primary antibody used for the six types of cells is Biotin-protein L. The secondary antibody used in both staining methods is PE-Streptavidin. In the first two rows of data,the experimental group for the primary antibody and fluorescent secondary antibody is represented as a solid black line,and control with only fluorescent secondary antibody is represented as sky blue shade. In the third row of data,the Jurkat cell with lentiviral infection group is represented as a solid black line and the 4 types of cells co-expressed by GFP and CAR are represented as sky blue shade. The X-axis represents the number of cells,and Y-axis represents the fluorescence intensity. The data shows the positive efficiency of CAR molecules

图5 两种染色方法平均荧光强度及阳性率比较 图4流式细胞仪检测结果量化得到平均荧光强度（每个样品值减去仅加荧光二抗的对照品数值）及阳性率,取3次独立重复实验均值进行分析

Fig. 5 Comparison of two staining methods shown as average fluorescence intensity and positive percentage The data measured by the flow cytometry in Fig. 4 are presented as the average fluorescence intensity（the value of each sample - the value of the control sample with only the fluorescent secondary antibody）and the positive percentage. The final data are the average of results from 3 independent experiments

图6 PCR片段DNA标准品的琼脂糖凝胶电泳图

Fig. 6 Agarose gel electrophoresis profile of the standard DNA samples by PCR

图7 标准品标准曲线图（绝对定量法） 取4个不同拷贝数梯度（标准品WPRE:1.23×103、1.23×105、1.23×107、1.23×109;标准品ALB:8.64×102、8.64×104、8.64×106、8.64×108）的标准品基因片段为模板进行荧光定量PCR。标准品计算所得的拷贝数进行以10为底的对数运算,得到的数值为横坐标,纵坐标为实时定量PCR结果中的循环阈值（Ct值）

Fig. 7 Standard curve diagrams of standard samples（absolute quantitative method） The gene fragments of standard samples with 4 different copy number gradients（standard WPRE:1.23×103,1.23×105,1.23×107,1.23×109;standard ALB:8.64×102,8.64×104,8.64106 and 8.64×108）as templates were in fluorescent quantitative PCR. The copy number of the standard samples was calculated with logarithmic based on 10,shown in X-axis,and the Y-axis is is the cycle threshold（Ct value）in the real-time quantitative PCR

图8 HL-GFP百分比与WPRE相对表达量的标准曲线图（相对定量法） 通过流式细胞仪读取Jurkat-Dupixent-HL-GFP绿色荧光阳性细胞比例。使用未感染的Jurkat细胞与Jurkat-Dupixent-HL-GFP绿色荧光阳性细胞混合,以获得GFP阳性梯度,分别为0（Jurkat）、20%、40%、60%。提取基因组进行qPCR检测,根据WPRE基因相对表达量（ALB基因为内参）和Jurkat-Dupixent-HL-GFP绿色荧光阳性的比例绘制标准曲线图

Fig. 8 Standard curve of the percentage of HL-GFP and the relative expression of WPRE（relative quantitative method） The percentage of Jurkat-Dupixent-HL-GFP positive cells was determined by flow cytometry. Uninfected Jurkat cells were mixed with Jurkat-Dupixent-HL-GFP positive cells to obtain the gradients of GFP positive cells:0（Jurkat）,20%,40% and 60%,respectively. The genome DNA was extracted for quantitative PCR detection,and a standard curve was depicted based on the relative expression of WPRE gene（ALB gene serves as an internal reference）and the percentage of Jurkat-Dupixent-HL-GFP positive population

图9 四种新型CAR分子3种不同检测方法的比较图 GFP共表达法为图4流式细胞仪检测结果中绿色荧光蛋白表达阳性率;相对定量法为图8相对定量标准曲线计算;绝对定量法为图7标准曲线计算得到平均每个细胞整合CAR分子的拷贝数

Fig. 9 Comparison of 3 different methods in detecting 4 novel CAR molecules The GFP co-expression refers to the percentage of GFP positive population measured by flow cytometry as shown in Fig. 4. The relative quantitative method refers to calculation via relative quantitative method as shown in Fig. 8. The absolute quantitative method refers to the average copy number of CAR molecules integrated per cell by substituting the standard curve of Fig. 7