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）

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

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

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

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

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

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

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

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