Figure 2.

Design and Characterization of a Chimeric Antigen Receptor Targeting ALK

(A) The ALK48 single-chain variable fragment was cloned into an MSGV1 retroviral expression vector containing a CD8α transmembrane-4-1BB-CD3ζ signaling motif to create the MSGV.ALK48.4-1BB.ζ construct encoding the ALK CAR. (B) Human peripheral blood mononuclear cells (PBMCs) were transduced with MSGV.ALK48.4-1BB.ζ CAR retroviral supernatant, and surface ALK CAR expression was evaluated by flow cytometry. (C) ALK CAR T cells were assayed for IFN-γ release after co-incubation with tumor targets. Differences in ALK CAR production of IFN-γ were evaluated by a one-way ANOVA followed by a Tukey’s multiple comparisons test, and they are representative of three experiments with different PBMC donors. (D) In vivo efficacy of ALK CAR T cells was assessed in two xenograft models of neuroblastoma. Growth curves of SY5Y or Kelly tumors after treatment with ALK CAR or mock-transduced T cells are shown. Differences in tumor growth were determined using a repeated-measures ANOVA. Error bars represent SEM (n = 5 mice per group [SY5Y] and n = 10 mice per group [Kelly]). In vivo experiments were repeated twice for each tumor model with different PBMC donors.