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. 2020 Jun 19;28(10):2237–2251. doi: 10.1016/j.ymthe.2020.06.022

Figure 1.

Figure 1

Screening of a Library of FLT3-Targeted scFvs and Selection of a High-Performing FLT3 CAR

(A) Activated primary human T cells were effectively transduced with lentiviral vectors encoding a range of FLT3 CARs. Bar graph shows the frequencies of transduced cells determined by flow cytometry analysis 3 days after transduction (mean ± SEM; n = 3 donors). (B) Target-independent “tonic” signaling was evaluated by measuring the frequency of transduced cells positive for the activation marker 4-1BB 6 days after transduction (mean ± SEM; n = 3 donors). (C) Higher tonic signaling observed with some scFvs (P1A5, P12B6, P14G2) was associated with a decrease in the TSCM cell subset indicative of increased CAR T cell differentiation. CAR T cells were analyzed by flow cytometry 9 days after transduction and phenotypes were determined according to CD62L and CD45RO expression within the CAR+ cell population: stem cell memory (CD45RO−/CD62L+), central memory (CD45RO+/CD62L+), effector memory (CD45RO+/CD62L−), and effector (CD45RO−/CD62L−) cells (mean ± SEM; n = 3 donors). (D) CAR T cells exhibited expansion upon repeated exposure to target. The proliferative capacity of CAR T cells was determined by counting viable CAR+ cells 20 days after co-culture with EOL-1 target cells. Fold-expansion is expressed as mean ± SEM relative to day 0; n = 2 donors. (E) Assessment of antitumor activity in an orthotopic model of AML led to effective differentiation of candidate scFvs and selection of the lead FLT3 CAR. NSG mice received a single dose of 2.5 × 106 or 5 × 106 FLT3 CAR T cells 10 days after injection of luciferase-labeled EOL-1 cells and tumor burden was assessed by bioluminescence imaging (BLI) over time (n = 10 mice/group). Values are expressed as mean ± SEM (∗∗∗∗p < 0.0001 by one-way ANOVA with Dunnett’s post hoc test for multiple comparisons versus non-transduced control T cells). Tumor growth kinetics for the P4C7 (2.5 × 106) and P9B5 (2.5 × 106) groups was comparable to control T cells, and their curves are not shown for simplicity.