Skip to main content
. Author manuscript; available in PMC: 2018 Dec 13.
Published in final edited form as: J Immunol. 2009 Nov 1;183(9):5563–5574. doi: 10.4049/jimmunol.0900447

FIGURE 3.

FIGURE 3.

Transgene decrease was correlated with loss of transduced cells from the culture and was associated with CD3ζ signaling in the CAR. A, A schematic representation of the 4D5-based CAR constructs, including scFv, hinge region (Hinge), transmembrane region (TM), CD28 intracellular domain (CD28 IC), CD3ζ intracellular domain (CD3 ζ IC), and the hinge and transmembrane region of CD8α (CD8). All of the 4D5 mutant scFvs and CD3ζ mutations were generated by PCR-based site-directed mutagenesis using the parent 4D5–28Z as template according to the published sequences (25). The published affinity of the different 4D5 Ab variants is: parent 4D5–28Z (3 × 10–10 M), 4D5–1 (2.5 × 10–8 M), 4D5–3 (4.4 × 10–9 M), 4D5–5 (1.1 × 10–9 M), and 4D5–7 (6.2 × 10–10 M). The vertical line indicates the approximate location of the mutation. B, Transgene expression over time after transduction. PBLs were transduced with 4D5–28Z-based CARs with scFv at different affinities (4D5–28Z, 4D5–1, 4D5–3, 4D5–5, 4D5–7), a 4D5 CAR with hinge and transmembrane regions from human CD8α without CD28 signaling domain (4D5-CD8HTZ), and a 4D5 CAR with both CD28 and CD3ζ signaling domains being truncated (4D5–28D). Construct organization was as depicted in A. Controls included a CAR against CD19 (FMC63–28Z) and an anti-NY-ESO-1 TCR (1G4-AIB). Transgene expression was monitored at days 8, 21, and 35 after transduction by flow cytometry staining of the transduced PBLs using gene-specific reagents. Data shown are percentages of day 8 transgene expression of each transduced PBL population. C, PBLs were transduced with 4D5-based CARs with various signaling domain alterations: 4D5 CAR with CD28 and CD3ζ signaling domains being truncated (4D5–28D), 4D5 CAR with all three ITAMs within CD3ζ being mutated (4D5–28ZM), CD28 signaling domain being truncated from 4D5–28Z (4D5–28HTZ), 4D5 CAR without CD28 signaling domain and hinge/transmembrane region from CD8α (4D5-CD8HTZ), and the parent 4D5–28Z (4D5–28Z). As controls, PBLs were transduced with anti-NY-ESO-1 TCR (1G4-AIB), LNGFR CAR (LNGFR-28Z), SP6 CAR (SP6–28Z), and a CD19 CAR (FMC63–28Z). All mutant constructs were as depicted in A. Transgene expression was detected at indicated time by flow cytometry for protein using gene-specific reagents (left panel), for RNA using real-time quantitative RT-PCR (middle panel), and for DNA copy number determined by real-time quantitative PCR (right panel). Data shown are percentages of day 8 transgene expression of each transduced PBL population. Data are representative of three experiments.