Figure 1. Construction and characterization of recombinant retroviral vectors expressing CARs targeted against mouse VEGFR-2.

(A) Schematic representation of recombinant retroviral vectors encoding CARs used in this study. In the DC101-CD828BBZ CAR vector, the DC101 ScFv is made up of the V_H and V_L derived from a rat IgG against mouse VEGFR-2 joined by a 218 linker that is linked to the hinge and transmembrane regions of the mouse CD8α chain and mouse intracellular signaling sequences derived from CD28, 4-1BB, and CD3-z molecules. The DC101-CD828Z vector lacked the 4-1BB signaling domain. The DC101-CD8 construct lacked all the intracellular signaling sequences. The SP6-CD828BBZ construct was derived from SP6, a mouse monoclonal antibody raised against a hapten 2, 4, 6-trinitrophenyl (TNP). (B) Enriched splenic CD3⁺ T cells were transduced with indicated retroviral vectors shown in A. Four days after transduction, cells were analyzed for transgene expression by flow cytometry. Representative FACS data are presented, showing the percentage of T cells in each quadrant, with the percentage of transgene-positive cells in parentheses. IsoAb, isotype control antibody. GAM, goat anti-mouse antibody. (C and D) Enriched CD3⁺ mouse T cells were transduced with the indicated retroviral vectors. Four days later, cells were cultured on antigen-coated 96-well microtiter plates for 3 days. (C) The proliferation of T cells was measured as [³H]thymidine incorporation during the last 16 hours. (D) Culture supernatants were assayed for IFN-γ by ELISA. Results are presented as the mean ± SEM of triplicates.