Figure 1.

ScFv optimization. (A) Schematic diagram showing the different anti-ROR1 Sc variants used for testing, including four different permutations of the fusion proteins Heavy (VH) and light (VL) chains with two different (G4s)₃ linkers - Long (L) and short (S). (B) Surface expression of anti-ROR1 specific CAR protein in primary T cells.1⁶ T cells were transduced with the different constructs using a lentiviral concentrate at a MOl 3.0. Expression was examined by flow cytometry at 6 days following transduction, using a ROR1-biotin soluble recombinant protein and streptavidin-PE. (C) CAR T cell expansion curve (D). After 14 days of expansion using a luciferase-based assay the different scFv configurations, the constructs were incubated with JeKo-1/ROR1+ and K662/ROR1- cells at an ET ratio of 10:1, and the cells were harvested at 24 hours. (E) After 24 hours stimulation with ROR1+ cells IFN- γ was measure by FACS. (F) Degranulation assay after 4 hours simulation with ROR1+ cells. Data are plotted as mean ‡ SEM (****, p < 0.00001, **, p < 0.001, and * p= < 0.01).