Extended Data Fig. 4: Coexpression of BCL-2 with dominant-negative receptors or combination with a single switch receptor transiently enhances antileukaemia activity of dual-CAR T cells.
From: Leucine zipper-based immunomagnetic purification of CAR T cells displaying multiple receptors
a, (Left) Diagram and maps for vectors encoding combinations of the Zip-sorting system, CD19 and CD20 dual-CAR, BCL-2 D34A caspase-cleavage resistant mutant, iC9, 3N-mutant blocked zipper-tagged PD-1-DNR (DNR; dominant-negative receptor), and FasDNR. 3N mutant blocking-zipper was used to increase affinity-tagged zipper surface expression (See Extended Data Fig. 1h). Tandem hCD20 mimotopes were added as expression tag and as a target for cell Rituximab-mediated depletion. (Right) Flow cytometry analysis of Zip-sorted BALB/c T cells dual-transduced with vectors as depicted in legend. Numbers in flow plots refer to post Zip-sort purity percentage for each marker. b, Maps for vector set encoding Q2-RR-iC9 and 3N zipper-tagged PD-1-CAR. c, Expression of PD-1-CAR and surface bound Q2-RR zipper. d, PD-L1 expression on BM185-CD20-PD-L1 clone. e, Luciferase-based 24 h target lysis assay using Zip-sorted Q2-RR-iC9/3N-EE-PD-1-CAR BALB/c T cells or non-transduced T cells. Data are mean ± SEM of triplicate wells for a representative experiment from n = 2 donor experiments. f–i, Sublethally irradiated BALB/c mice were injected with a 1:1 mixture of BM185-CD19 and BM185-CD20 and treated with Zip-sorted CD45.1+ congenic BALB/c T cells. f, Leukaemia BLI (ffluc) from single experiment. g, Flow cytometry analysis of CAR T cells in peripheral blood on day 9. h, Linear regression analysis of leukaemia BLI signal vs. blood CD45.1+ T cell concentration. i, Survival. j–l, Experimental setup as in panel f, but mice were treated with dual-CAR T cells incorporating FasBB switch receptor instead of FasDNR. j, Leukaemia BLI from three combined experiments. k, Flow cytometry analysis of CAR T cells in peripheral blood on day 10, combined from two experiments. l, Survival from three combined experiments. Log-transformed BLI AUC values were compared using a Vardi test with FDR correction. Blood T cell counts were compared with a two-tailed t-test. Survival was compared with log-rank or pairwise log-rank comparison with FDR correction.
