1. A, RT-qPCR of ROR1 mRNA expression in B-CLL cells and a panel of human and rhesus macaque tissues.
A, RT-qPCR of ROR1 mRNA expression in B-CLL cells and a panel of human and rhesus macaque tissues. ROR1 expression was normalized to the geometric mean of the housekeeping genes GAPDH and TBP. B-CLL sample #1 was used as the reference control (ROR1 expression = 1). B, cytolytic activity of rhesus T cells modified to express a R12-ROR1 CAR with human (hu) or rhesus macaque (rh) 4-1BB and CD3ζ domains against 51Cr-labeled K562/ROR1 or K562 cells. Results (mean, SEM) are from three independent experiments in 3 animals (A11047, A13011, and A13002). C, flow cytometric analysis of purity and phenotype of ROR1 CAR-modified CD4+ and CD8+ T cells showing expression of tCD19 in CD4+ (top) or CD8+ (bottom) T cells either mock-transduced, after transduction with the ROR1 CAR (preselection), after selection for tCD19 expression (postselection 1), and after selection for CD4+ or CD8+ cells, respectively (postselection 2). All samples are gated on CD3+ cells. D, cytolytic activity of CD4+ and CD8+ ROR1 CAR-T cells against 51Cr-labeled K562/ROR1 or K562 cells. E, proliferation of CFSE-labeled ROR1 CAR-T cells 72 hours after stimulation with K562/ROR1 cells or unmodified K562 cells. CFSE-dye dilution is shown after gating on CD3+CD4+tCD19+ or CD3+CD8+tCD19+ T cells. F, Luminex cytokine assay of supernatants obtained after 24 hours from triplicate cocultures of 5 × 104 CD4+ or CD8+ ROR1 CAR-T cells with K562/ROR1 cells or media alone as described in Materials and Methods. The asterisk (*) denotes cytokine levels below detection level. Cytokine levels in media controls were below detection level. Data in C to F are shown for macaque A13002 and are representative of results in 3 animals.
Carolina Berger et al. Cancer Immunol Res 2015;3:
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