PMC

(A) ScFv titration to assess binding to K562/CD19 cells. (B) Competition binding assay of each scFv with FMC63. Binding of FMC63 and competition with itself (open symbols) are shown as a comparison in each graph.

(A) Flow cytometric analysis of PD-1 expression on CD19-CAR-T-cells ten days after re-stimulation with CD19⁺ LCL. (B,C) Fluorescence microscopy images of T-cells expressing CD19-CAR-eGFP fusion proteins in the absence (B) or presence (C) of Hoechst33342-labeled (blue) Raji cells. Data in A–C are representative of at least two experiments.

(A,B,C) Western Blot analysis of CAR protein (~50 kD) based on CD247 staining. Endogenous CD247 (18 kD) was used as a loading control. Data are representative of at least two experiments. (D) Sequences of human and rhesus macaque CD19 are shown for aa 218–288. Aa that are different are marked in the human sequence. (E) IFN-γ concentrations in supernatants of CD19-CAR-T-cells after co-culture with K562 cells expressing either rhesus macaque, human, or chimeric versions of CD19. (F) IFN-γ concentrations in supernatants of CD19-CAR-T-cells after co-culture with K562 cells expressing either rhesus macaque or human CD19 with single or double mutations to the other species. Data in E and F are representative of at least two experiments.

(A) Predicted affinities to MHC I of 9-mer peptides derived from the CD28/4-1BB fusion site. Reciprocals of predicted affinities in nM were plotted for all tested MHC types. A predicted affinity of <100 nM (>0.01) was chosen as a threshold. Peptides above the threshold and corresponding HLA types are specified. (B) Cytolytic activity, (C) IFN-γ production, and (D) proliferation of CD8⁺ T-cells expressing CARs with either the original or the new CD28/4-1BB fusion site after co-culture with K562, K562/CD19, and Raji cells. Data in B–D are representative of at least two experiments with T-cells prepared from different donors.

(A) Bioluminescence imaging of Raji/ffluc tumor growth in NSG mice that received 5x10⁶ (4 mice per group) and 1x10⁶ (9 mice per group) CD8⁺ CD19-CAR-T-cells, respectively. EGFRt-T-cells were used as control (4 mice per group). Arrows mark the day of T-cell transfer. (B) Cytokine concentrations in supernatants of CD4⁺ CD19-CAR-T-cells after stimulation with K562, K562/CD19, and Raji cells for 24 h. (C) Proliferation of CD4⁺ CD19-CAR-T-cells after stimulation with K562, K562/CD19, and Raji cells for 72 h. Data in B and C are representative of three experiments with T-cells prepared from different donors. (D) Bioluminescence imaging of Raji/ffluc tumor growth in NSG mice (5 mice per group) that received a mixture of 5x10⁵ CD4⁺ and 5x10⁵ CD8⁺ CAR-T-cells. EGFRt-T-cells were used as a control (4 mice per group). The arrow marks the day of T-cell transfer.

(A) EGFRt expression on CD8⁺ T-cells transduced with CD19-CAR-EGFRt constructs before enrichment (pre) and after enrichment and expansion (post). (B) Cytolytic activity of EGFRt⁺ CD19-CAR-T-cells against CD19⁺ (K562/CD19, Raji) and control (K562) target cells analyzed by a 4 h chromium release assay at an E:T ratio of 30:1. (C) IFN-γ concentrations in supernatants of CD19-CAR-T-cells after stimulation with K562, K562/CD19, and Raji cells for 24 h analyzed by ELISA. Data in A-C are representative of at least five experiments with T-cells prepared from different donors. Differences between HL and LH constructs are significant (p<0.05) for each CAR. (D) Proliferation of CD19-CAR-T-cells after stimulation with K562, K562/CD19, and Raji cells for 72 h analyzed by a CFSE dilution assay. Data are representative of at least three experiments with T-cells prepared from different donors. (E) Cytolytic activity, IFN-γ production, and proliferation of CD19-CAR-T-cells after co-culture with primary CLL or K562 cells. Data for cytolytic activity and IFN-γ production are shown as means of three CLL samples isolated from different patients. Proliferation data are representative for three CLL samples.