Figure 4.

In vitro validation of CAT/9A8

(A) CAT and 9A8 were expressed on a separate lentiviral pCCL vectors driven by a PGK and EF1α promoter, respectively. (B) Lentiviral vectors produced transiently in 293T were mixed 1:1 to transduce primary T cells (MOI = 2.5 + 2.5). The CAT/9A8 product constitutes the expression of both CAT and 9A8 CARs at a stochastic mix. (C) The CAR expression was measured by the labeling of T cells with anti-CAT idiotype and sCD22 antigen as shown in x- and y axes, respectively. The mix of the vectors leads to a profile of three transduced populations of Single^CAT, Single^9A8 and double-positive. We cultured 5 × 10⁴ SupT1 cells engineered to express CD22^High, CD22^Mid, CD19^High, or CD19^HighCD22^High with CAR T cells at a ratio of 1:8 for 72 h. At the endpoint of this assay, the target cell lysis (D) and IFN-γ release (E) were assessed. (F) The efficacy of the CAT/9A8 product was also tested against the physiological Raji WT cells and Raji CD19^KO, which was implemented to simulate CD19 antigen escape. Student's t-test was utilized to determine statistical significance (n = 10).