Figure 5.

Construction of dual-specific CARs

(A) Schematic representation of lentiviral CAR construct containing a deimmunized anti-TAG-72 scFv, CD8 hinge domain, CD8 or CD28 TM domain, and either a CD28 or 4-1BB intracellular signaling domain linked to a CD3ζ signaling domain coupled to an anti-CD47 scFv, CD28 hinge domain, CD28 TM domain, and truncated intracellular domain resulting in a non-signaling dimerizing CAR (ΔCD47d). (B) Flow cytometric analysis was performed to ascertain frequency of CAR and CD3 co-expression following transduction (n = 5–8). (C and D) CAR-T cell expansion (C) was tracked for 11 days after transduction, and viability (D) was monitored following 7 days of expansion. (E) Schematic showing constructs similar to those of (A) but with an anti-CD47 scFv and Cys-to-Ser point mutations within the CD28 hinge and TM domain resulting in a non-signaling, monomeric CAR (ΔCD47m). (F) Schematic representation of CAR localization at the cell membrane depicting the difference in TAG-72 CAR + ΔCD47d CAR (left) and TAG-72 CAR + ΔCD47m CAR (right). (G) Western blot analysis of TAG-72 + ΔCD47 dual CAR-T cell lysates in the presence or absence of β-mercaptoethanol (β-met), with reducing (+) and non-reducing (–) conditions. ΔCD47 CAR bands were detected by anti-c-Myc antibody, and TAG-72 CAR bands were detected by anti-CD3ζ antibody. Molecular mass markers (M) are indicated to the right of each panel. (H and I) Transduction efficiency (H) and expansion potential (I) were monitored as previously described. (J) Total viable cells following 11 days of expansion were directly compared for TAG-72.CD28 dual CAR-T cells (left) and TAG-72.4-1BB dual CAR-T cells (right). Data represent average ± SD of biological replicates generated in independent experiments. n.s. not significant, ∗p < 0.05, ∗∗∗p < 0.001.