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. 2018 Nov 19;9:2268. doi: 10.3389/fimmu.2018.02268

Figure 1.

Figure 1

TAG72-specific CAR T cells containing a 4-1BB intracellular co-stimulatory domain. (A) Diagram of the lentiviral expression cassette with TAG72-CARs containing the humanized scFv (CC49 clone) targeting TAG72, with a 129 amino acid modified human IgG4 Fc linker (void of the CH2 domain, ΔCH2), a CD4 transmembrane domain, a cytoplasmic 4-1BB costimulatory domain, and a cytolytic CD3ζ domain. A truncated non-signaling CD19 (CD19t), separated from the CAR sequence by a T2A ribosomal skip sequence, was expressed for identifying lentivirally transduced T cells. (B) Mock (untransduced) and TAG72-BBζ CAR T cells were evaluated by flow cytometry for CD19t expression to detect lentiviral transduction of CARs (left) or Protein L to detect the scFv (right). (C) CD4 and CD8 expression in Mock (top) and TAG72-BBζ CAR T cells (bottom). (D) Activation (expression of CD137) was assessed by flow cytometry with in vitro stimulated CAR T cells against soluble or plate-bound purified TAG72 antigen for 24 h at indicated protein amounts.